UCSD Human Research Protections Program New Biomedical Application RESEARCH PLAN Instructions for completing the Research Plan are available on the HRPP website. The headings on this set of instructions correspond to the headings of the Research Plan. General Instructions: Enter a response for all topic headings. Enter “Not Applicable” rather than leaving an item blank if the item does not apply to this project. Version date: 9/30/2013 1. PROJECT TITLE Measuring Human Brain Activity during repetitive Transcranial Magnetic Stimulation in healthy participants 2. PRINCIPAL INVESTIGATOR Dr. Scott Makeig, Director, Swartz Center for Computational Neuroscience (SCCN), Institute for Neural Computation, UCSD 3. FACILITIES - UCSD, Swartz Center for Computational Neuroscience, Institute for Neural Computation - UCSD, PrTMS Research Center, 16918 Dove Canyon Rd (Suite 100), San Diego, CA 92127 4. ESTIMATED DURATION OF THE STUDY The study is estimated to run for 2 years from a proposed start date of March 1st 2018. 5. LAY LANGUAGE SUMMARY OR SYNOPSIS (no more than one paragraph) Repetitive Transcranial Magnetic Stimulation (rTMS) is a method for regionally blocking or facilitating cortical processes by stimulating the human brain noninvasively. In single pulse mode TMS, a coil is placed over the surface of the head and current is passed through the coil very quickly. A very small current is induced in the underlying brain via magnetic transduction, fairly focal to the coil. For example, doing this over the primary motor cortex can produce a slight twitch in the connected muscles. Low-frequency (once per second) TMS induces a transient suppression of excitability in the affected cortical region(s), while faster rTMS at stimulation frequencies of 5 Hz and higher transiently enhances local cortical excitability. Recently it has been suggested that the behavioral enhancement produced by faster rTMS is mediated by affecting endogenous oscillations in cortical field potentials. This project aims to investigate the interaction of excitatory rTMS (at a frequency near 10 Hz selected to match the subject’s natural brain activity frequencies) and their ongoing oscillatory activity in the human cortex by using simultaneous electroencephalography (EEG). EEG is a safe and commonly used method for measuring electrical activity in the brain from the surface of the scalp. 6. SPECIFIC AIMS This protocol will examine the effects of rTMS on oscillatory activity in the human cortex using electroencephalography (EEG). High-density EEG will be recorded before, during and after rTMS stimulation at frequencies near 10 Hz over frontal cortex. The EEG data will be recorded from healthy participants at the PrTMS Research Center. Healthy participants will undergo one session with simultaneous EEG/rTMS. The PrTMS Research Center has acquired a new TMS compatible EEG amplifier (BrainVision actiCHamp, Brain Products, Germany) that reduces TMS artifacts in the EEG and records at better than 1-ms precision. This allows examining relationships between single rTMS pulses and ongoing oscillatory EEG activity. This setup will allow us to examine changes in cortical dynamics occurring during as well as following stimulation bursts as well as changes in resting state networks between stimulation periods. To date, the effects of rTMS on cortical oscillations have not been well understood. Using advanced analysis methods for EEG source separation and localization we will examine the following temporally and spatially short and long range effects of rTMS on cortical activity. The study will in particular seek to answer the following questions: 1) How does rTMS near 10 Hz affect endogenous neuronal oscillations? 2) How does rTMS near 10 Hz affect excitatory/inhibitory networks in the cortex? 3) How are these effects on neuronal oscillations and/or excitatory/inhibitory networks related to observable behavioral changes? 4) 5) 6) 7) How does rTMS affect activity at cortical areas distant from the stimulation site(s)? How does the rTMS stimulation affect functional connectivity between brain areas in the EEG signals? How do parameters of stimulation such as stimulator intensity and frequency affect the prior points? How does the brain state at the time of stimulation (i.e. the phase of ongoing alpha at the time of the TMS pulse) affect the changes induced by rTMS? Brain stimulation protocols and treatments are on the rise and are increasingly used to treat neurological and mental disorders, however, stimulation parameters are often arbitrarily defined. It is therefore imperative to investigate how these parameters influence the effects of rTMS and scientifically define stimulation parameters. 7. BACKGROUND AND SIGNIFICANCE Repetitive TMS. Transcranial magnetic stimulation (TMS) offers a noninvasive method of inducing electrical activation of the brain. Repetitive facilitatory high-frequency stimulation (≥ 5 Hz) TMS (dubbed rTMS), delivers grouped pulses of stimulation at precise frequencies and has been shown to interact with endogenous neuronal oscillations in the human cortex (Klimesch et al, 2003, Thut & Miniussi, 2009, Thut et al, 2011; Veniero et al, 2011; Froehlich, 2015). Single pulses of TMS produce complex but short responses. Repeated pulses can have more prolonged effects on the brain. rTMS over a cortical site leads to effects on metabolic activity that outlast the period of stimulation (Thut & Pascual-Leone, 2009). These effects of rTMS can be observed not only at the local site of stimulation but also at remote, anatomically and functionally connected cortical sites (Fuggetta et al, 2008). Some of these after effects might depend on long term potentiation (LTP) and cortical depressionlike changes in synaptic connections between cortical neurons (Ridding & Rothwell, 2007). There is also good evidence that the changes resulting from rTMS influence natural behaviors (Hamidi et al, 2009). The longlasting neuromodulatory effects of repetitive transcranial magnetic stimulation (rTMS) are of great interest for therapeutic applications in various neurological and psychiatric disorders, in which functional connectivity among brain regions is disturbed (Fuggetta & Noh, 2013; Ding et al, 2014; Jin et al, 2005). EEG brain imaging. For the last two decades we (Dr. Makeig and colleagues) have been developing, testing, and demonstrating methods for resolving, locating, and modeling the dynamics of high-density electroencephalographic (EEG) signals recorded non-invasively from the human scalp. Key tools for this are Independent Component Analysis (ICA) (Makeig et al., 1996) and inverse source modeling (Makeig et al., 2002; Delorme et al., 2011; Akalin Acar et al., 2016); we have shown these two methods are complementary and give, for the first time, the possibility of tracking the continuous local field potential signals from up to dozens of cortical areas with relatively high stimulus-noise ratio (SNR). In this project we propose to further advance, test, validate and apply the resulting advances in the power of functional EEG brain imaging to investigate the effect of rTMS on the ongoing EEG source activity and connectivity in resting state EEG which is of interest to basic and clinical cognitive neuroscience. Measuring the neuromodulatory effects of TMS. A variety of methods have been introduced to measure the neuromodulatory effects of TMS. Electromyography (EMG) has been mainly used with single pulse TMS to investigate normal and impaired motor cortical excitability. Stimulating over the motor cortex introduces an activation of a hand muscle for example, and the amplitude of the resulting muscle activation can be used as a measure of motor cortical excitability. The major drawback of the TMS-EMG protocol is that the intervention and read-out is largely limited to the motor cortex (M1) and its descending direct pathways to the muscle. EEG and TMS. Recent developments that combine TMS and functional neuroimaging techniques, including electroencephalography (EEG) functional magnetic resonance imaging (fMRI) and positron emission tomography (PET), have extended the investigation of modulation of cortical excitability to regions other than M1. Neuroimaging techniques have a great potential to map spatiotemporal patterns of functional reorganization that are induced in the human brain by rTMS. Biomedical IRB Application Instructions Page 2 TMS may be applied ‘‘offline’’ before or after neuroimaging, or ‘online’ while neuroimaging is being performed. The ‘‘offline’’ TMS-neuroimaging approach is technically easy to establish because rTMS and neuroimaging are separated in time. ‘‘Offline’’ TMS-neuroimaging after conditioning with rTMS can be used to map brain areas and brain networks that show persistently altered activity during subsequent (1) resting-state (e.g., due to metabolic changes), or (2) task performance (e.g., due to rapid reorganization). Online TMS-neuroimaging, on the other hand, is technically demanding because TMS may adversely affect data acquisition during neuroimaging. The EEG method is a perfect complement for the online TMS approach since its high-temporal resolution in the millisecond-scale allows to measure the immediate cortical response to TMS. In past setups the very immediate cortical response to TMS within the first 10 or 20 msec after TMS stimulation was still not fully accessible because of the TMS induced artifact in the EEG. The new amplifier (BrainAmp, Brain Products) that the PrTMS Research Center acquired, reduces the TMS artefact below 1 ms. Using concurrent source separation approaches with ICA to get rid of residual muscle artefacts on the scalp we can now directly access the EEG within 2 ms after the TMS pulse. This will allow us to examine the very immediate cortical response to TMS and subsequent pulses during rTMS. The online TMS-EEG approach will allow us: 1) to study the state dependency of the brain’s responsiveness to TMS by looking at EEG activity just before a TMS stimulus is applied. The regional expression of spontaneous oscillatory activity directly preceding a TMS pulse may be predictive of the brain response to TMS; 2) to reveal TMS induced changes in the frequency domain. For instance, a single TMS pulse can transiently synchronize activity in the beta range of the EEG. More generally, TMS-EEG will allow us to prospectively track and monitor the excitability and connectivity changes occurring in any cortical region during rTMS. The online TMS-EEG approach will allow us also to systematically investigate effects of stimulation intensity and frequency on endogenous oscillations in the EEG and relate interactions between cortical oscillations and rTMS to changes in overt behavior. Functional effects of 10-Hz rTMS The effects of rTMS have been investigated in two different timescales: immediate and longer-lasting effects. Here we will mainly focus on immediate effects of rTMS during the stimulation (resulting from direct neuronal excitation/inhibition and interaction with ongoing brain activity) and short term after effects of rTMS from 1 sec to 20 min after the stimulation. Effects based on oscillatory entrainment: It has been shown that rTMS results in phase-coupling (also termed entrainment) between oscillatory brain activity and the external electromagnetic stimulus. In terms of immediate effects during rTMS, there are multiple examples that this approach may indeed lend itself for a targeted intervention into oscillatory brain activity through entrainment that can affect brain function and behavior using rTMS. However, most of the evidence for the existence of entrainment effects comes from behavioral studies. In contrast, only very few rTMS studies so far have managed to simultaneously record EEG (due to contamination of the recorded neurophysiological signals by stimulation-induced artifacts). Even fewer have combined the two, i.e. recorded EEG and documented the associated behavioral effects. However, online registration of the EEG signal is required to verify rTMS interaction with brain oscillations (here entrainment) as the basis of the behavioral change. Some of the behavioral and offline EEG studies indirectly support entrainment by frequency-tuned rTMS. When rTMS is frequency-tuned to known, task-related oscillations, (for example alpha band oscillations), associated behavioral performance measures and offline EEG power spectra are biased in expected directions, i.e., in line with known correlative brain-behavior relationships (Klimesch et al., 2003; Veniero et al, 2011, Thut et al, 2011), suggesting that rTMS interacts selectively with the target oscillations and associated function by synchronization. For example Klimesch et al 2013 showed that alpha power increase after 10-Hz rTMS was related to a significant improvement in performance in a mental rotation task. Biomedical IRB Application Instructions Page 3 Effects based on suppression of inhibitory networks: Evidence has been provided that 10-Hz rTMS is capable of modulating inhibitory networks. A reduction in inhibitory neurotransmission may facilitate the expression of associative plasticity in cortical networks. These studies (in mice, Lenz et al, 2016) have suggested that 10-Hz rTMS may act by decreasing GABAergic neurotransmission onto cortical principal neurons. These findings support a model in which rTMS-induced long-term depression (LTD) of GABAergic synaptic strength mediates changes in excitation/inhibition-balance of cortical networks, which may in turn facilitate (or restore) the ability of stimulated networks to express input-and task-specific associative synaptic plasticity. 10-Hz rTMS may therefore promote associative learning and memory through induction of plasticity by depression of local inhibitory networks. How can we measure GABAergic neurotransmission non-invasively? In humans it is possible to directly measure GABAergic Neurotransmission in the Human Cortex and assess human cortical excitability and connectivity by combining transcranial magnetic stimulation (TMS) and electroencephalography (EEG) (Premoli et al, 2014; 2014; Rogasch et al, 2013). TMS of the primary motor cortex elicits a sequence of TMSevoked EEG potentials (TEPs). It is thought that inhibitory neuro-transmission through GABA-A receptors (GABAAR) modulates early TEPs (50 ms after TMS), whereas GABA-B receptors (GABABR) play a role for later TEPs (at 100 ms after TMS) (Premoli et al, 2014). Furthermore paired-pulse transcranial magnetic stimulation (TMS) protocol of long-interval intracortical inhibition (LICI) combined with electroencephalography is a method for studying cortical inhibition by assessing GABAB-mediated cortical inhibition (Rogasch et al., 2013). A single suprathreshold TMS pulse activates all Interneurons. We see activation in the muscle of the contralateral hand because the excitatory interneurons activate the pyramidal neurons, sending a descending volley to the spinal cord, then to the muscle. Inhibitory interneurons have a lower threshold than facilitatory ones. This means that a weak, subthreshold TMS pulse will selectively activate the inhibitory Figure1: Inhibitory and excitatory interneurons (left) and their activation by paired pulse TMS (right). interneurons. (Note: In this case we don’t see muscle activation in the hand because the excitatory interneurons haven’t activated the pyramidal neurons). When a subthreshold TMS pulse is followed 1 – 200 ms later by a suprathreshold TMS pulse, the first pulse activates inhibitory interneurons which release GABA. This activates GABA receptors on the excitatory interneurons, so when the second pulse (suprathreshold) occurs, the excitatory interneurons are less able to be activated This results in fewer excitatory interneurons firing, and fewer pyramidal neurons firing, and a smaller muscle activation. Recently, first studies using a combination of TMS and electroencephalography (TMS–EEG) have started to directly investigate mechanisms of LICI at the cortical level (Daskalakis et al., 2008; Farzan et al., 2010; Rogasch et al., 2013). It was shown that a conditioning stimulus (LICI protocol) induced a reduction of the Biomedical IRB Application Instructions Page 4 averaged TS-evoked EEG potential (TEP) recorded at the C3 electrode, which is located above the stimulated M1 (Daskalakis et al., 2008). A recent study has shown that it is possible to study cortical inhibition in the dorsolateral prefrontal cortex (DLPFC) (Rogasch et al, 2015). The authors used single and paired pulse (interstimulus interval, 100 msec) TMS to the left DLPFC to assess the relationship between distinct TEPs and oscillations and investigate the relationship of these mechanisms to working memory. The results suggest that both the LICI paradigm and N100 following single pulse TMS reflect complementary methods for assessing GABAB-mediated cortical inhibition in the DLPFC. We will use single and paired pulse TMS in this protocol to assess whether 10-Hz rTMS is depressing of GABA receptors in the human cortex (similar to results in animal studies). Investigation of the effect of rTMS stimulator output intensity on the brain. One important aspect of TMS stimulation is the intensity of TMS pulses. As we have discussed in the previous paragraph depending on the intensity of the TMS pulse stimulation can have inhibitory or excitatory effects. Intensity of rTMS is measured according to the Resting Motor Threshold. The Resting Motor Threshold (RMT) is measured by stimulating over the right primary motor cortex area with single TMS pulses at successively increasing levels until a muscle twitch is produced at the thumb of the left hand. The RMT is evaluated for every person individually. rTMS studies and treatment protocols have typically used stimulation intensities above motor threshold. However recently studies have shown behavioral changes caused by rTMS also at stimulation intensities below motor threshold. An advantage of lower stimulation intensities is the reduced discomfort experienced by participants and the reduced risks associated with high frequency rTMS. On the other hand, it is important to make sure that rTMS stimulation intensity is high enough to produce an observable effect in behavior and cortical oscillations. Research has shown that there is an observable cortical effect of stimulation at levels below the RMT. Kaekoehnen et al, 2005 showed that left prefrontal focal TMS at intensities of 60%, 80%, 100%, and 120% of the motor threshold evoked clear responses in the EEG at all intensities. Although the response amplitude increases with stimulus intensity, scalp distributions of the potential are relatively similar for the four intensities (Komssi, et al, 2004). The results imply that TMS is able to evoke measurable brain activity at low stimulus intensities, probably significantly below 60% of RMT. Huang et al 2004, applied five or 15 pulses of 50-Hz rTMS at 50–80% active motor threshold (AMT). Results showed increased excitability/ facilitatory effect at 70 and 80% of ATM but not at 50%. Fuggetta et al, 2008 showed that subthreshold rTMS at alpha and beta frequencies produced a transient increase in power in the respective frequency band 2-4s after the stimulation train. In addition the authors found that subthreshold rTMS affected alpha-band activity more than threshold rTMS, inducing a specific decrease in alpha synchrony over the posterior regions during the trains of stimulation. Other studies suggest that the effects of rTMS may be also dependent on stimulation intensity. Doeltgen et al. (2007), for example, showed that 5-Hz subthreshold rTMS at 65% of RMT has an inhibiting effect while subthreshold rTMS at 70% RMT has a facilitatory effect. Given this prior evidence there are two main reasons to systematically investigate the effect of 10-Hz rTMS stimulation intensity on behavior, cortical patterns and cortical excitability. 1) It is not clear whether stimulation at different intensities may have opposite or diverse effects on cortical excitability and 2) if stimulation at very low intensities such as 60% or 40% would produce behavioral and/or cortical effects, parameters for treatments with rTMS could be adjusted to these low intensities making treatments better tolerable, less invasive and reducing the risks for adverse effects. Software tool development. Efficient use of the new brain imaging technology for studying human brain function requires development of new analytic tools capable of decomposing both EEG data into activities of functionally distinct brain systems and extra-brain (noise) sources. Our freely-distributed EEGLAB environment (Delorme & Makeig, 2004) is now by a recent survey the most widely used software environment for EEG signal processing by cognitive neuroscientists. Continuing advances in software tools require data Biomedical IRB Application Instructions Page 5 from specially designed EEG and other experiments. Adequate demonstrations of the power of our tools require experimental conditions involving a range of cognitive states and activities, performed by us and/or in collaboration with other cognitive neuroscience researchers. References: (1) Bell, AJ, & Sejnowski, TJ. (1995). An information-maximization approach to blind separation and blind deconvolution. Neural Comput, 7:1129-59. (2) Bystritsky, A., Kaplan, J.T., Feusner, J.D., Kerwin, L.E., Wadekar, M., Burock, M., Wu, A.D. & Lacoboni, M. (2008). A preliminary study of fMRI-guided rTMS in the treatment of generalized anxiety disorder. Journal of Clinical Psychiatry, 69(7), pp.1092-1098. (3) Daskalakis, Z. J., Farzan, F., Barr, M. S., Maller, J. J., Chen, R., & Fitzgerald, P. B. (2008). Long-interval cortical inhibition from the dorsolateral prefrontal cortex: a TMS–EEG study. Neuropsychopharmacology, 33(12), 2860-2869. (4) Delorme, A., Mullen, T., Kothe, C., Acar, Z. A., Bigdely-Shamlo, N., Vankov, A., & Makeig, S. (2011). EEGLAB, SIFT, NFT, BCILAB, and ERICA: new tools for advanced EEG processing. Computational intelligence and neuroscience, 2011, 10. (5) Diefenbach, GJ, Bragdon, LB, Goethe, JW. (2013) Treating anxious depression using repetitive transcranial magnetic stimulation. J Affect Disord; 151: 365–8. (6) Ding, L., Shou, G., Yuan, H., Urbano, D., & Cha, Y. H. (2014). Lasting modulation effects of rTMS on neural activity and connectivity as revealed by resting-state EEG. IEEE Transactions on Biomedical Engineering, 61(7), 2070-2080. (7) Doeltgen, S. H., & Ridding, M. C. (2011). Low-intensity, short-interval theta burst stimulation modulates excitatory but not inhibitory motor networks. Clinical Neurophysiology, 122(7), 1411-1416. (8) Farzan, F., Barr, M. S., Levinson, A. J., Chen, R., Wong, W., Fitzgerald, P. B., & Daskalakis, Z. J. (2010). Reliability of long-interval cortical inhibition in healthy human subjects: a TMS–EEG study. Journal of neurophysiology, 104(3), 1339-1346. (9) Fröhlich, F. (2015). Experiments and models of cortical oscillations as a target for noninvasive brain stimulation. Progress in brain research, 222, 41-73. (10) Fuggetta, G., Pavone, E. F., Fiaschi, A., & Manganotti, P. (2008). Acute modulation of cortical oscillatory activities during short trains of high-frequency repetitive transcranial magnetic stimulation of the human motor cortex: A combined EEG and TMS study. Human brain mapping, 29(1), 1-13. (11) Fuggetta, G., & Noh, N. A. (2013). A neurophysiological insight into the potential link between transcranial magnetic stimulation, thalamocortical dysrhythmia and neuropsychiatric disorders. Experimental neurology, 245, 87-95. (12) Hamidi, M., Slagter, H. A., Tononi, G., & Postle, B. R. (2009). Repetitive transcranial magnetic stimulation affects behavior by biasing endogenous cortical oscillations. Frontiers in integrative neuroscience, 3, 14. (13) Kähkönen, S., Komssi, S., Wilenius, J., & Ilmoniemi, R. J. (2005). Prefrontal transcranial magnetic stimulation produces intensity-dependent EEG responses in humans. Neuroimage, 24(4), 955-960. (14) Klimesch, W., Sauseng, P., & Gerloff, C. (2003). Enhancing cognitive performance with repetitive transcranial magnetic stimulation at human individual alpha frequency. European Journal of Neuroscience, 17(5), 1129-1133. Biomedical IRB Application Instructions Page 6 (15) Komssi, S., Kähkönen, S., & Ilmoniemi, R. J. (2004). The effect of stimulus intensity on brain responses evoked by transcranial magnetic stimulation. Human brain mapping, 21(3), 154-164. (16) Lenz, M., Galanis, C., Müller-Dahlhaus, F., Opitz, A., Wierenga, C.J., Szabó, G., Ziemann, U., Deller, T., Funke, K. and Vlachos, A., 2016. Repetitive magnetic stimulation induces plasticity of inhibitory synapses. Nature communications, 7, p.10020. (17) Lenz, M., & Vlachos, A. (2016). Releasing the cortical brake by non-invasive electromagnetic stimulation? rTMS induces LTD of GABAergic neurotransmission. Frontiers in neural circuits, 10. (18) Makeig, S, Bell, AJ, Jung, TP, Sejnowski, TJ. (1996). Independent Component Analysis of electroencephalographic data. In: D. Touretzky, M. Mozer, and M Hasselmo (Eds), Advances in Neural Information Processing Systems 8, 145-151. (19) Makeig, S, Westerfield, M, Jung, TP, Enghoff, S, Townsend, J, Courchesne, E, Sejnowski, TJ. (2002). Dynamic brain sources of visual evoked responses. Science, 295: 690-693. (20) Premoli, I., Castellanos, N., Rivolta, D., Belardinelli, P., Bajo, R., Zipser, C., Espenhahn, S., Heidegger, T., Müller-Dahlhaus, F. and Ziemann, U., 2014. TMS-EEG signatures of GABAergic neurotransmission in the human cortex. Journal of Neuroscience, 34(16), pp.5603-5612. (21) Premoli, I., Rivolta, D., Espenhahn, S., Castellanos, N., Belardinelli, P., Ziemann, U. and Müller-Dahlhaus, F., 2014. Characterization of GABAB-receptor mediated neurotransmission in the human cortex by pairedpulse TMS–EEG. Neuroimage, 103, pp.152-162. (22) Ridding, M. C., & Rothwell, J. C. (2007). Is there a future for therapeutic use of transcranial magnetic stimulation?. Nature Reviews Neuroscience, 8(7), 559-567. (23) Rogasch, N. C., Daskalakis, Z. J., & Fitzgerald, P. B. (2013). Mechanisms underlying long-interval cortical inhibition in the human motor cortex: a TMS-EEG study. Journal of Neurophysiology, 109(1), 89-98. (24) Rogasch, N. C., Daskalakis, Z. J., & Fitzgerald, P. B. (2015). Cortical inhibition of distinct mechanisms in the dorsolateral prefrontal cortex is related to working memory performance: a TMS–EEG study. Cortex, 64, 68-77. (25) Thut, G., & Pascual-Leone, A. (2010). A review of combined TMS-EEG studies to characterize lasting effects of repetitive TMS and assess their usefulness in cognitive and clinical neuroscience. Brain topography, 22(4), 219. (26) Thut, G., & Miniussi, C. (2009). New insights into rhythmic brain activity from TMS–EEG studies. Trends in cognitive sciences, 13(4), 182-189. (27) Thut, G., Veniero, D., Romei, V., Miniussi, C., Schyns, P., & Gross, J. (2011). Rhythmic TMS causes local entrainment of natural oscillatory signatures. Current biology, 21(14), 1176-1185. (28) Veniero, D., Brignani, D., Thut, G., & Miniussi, C. (2011). Alpha-generation as basic response-signature to transcranial magnetic stimulation (TMS) targeting the human resting motor cortex: A TMS/EEG co-registration study. Psychophysiology, 48(10), 1381-1389. (29) Jin, Y., Potkin, S. G., Kemp, A. S., Huerta, S. T., Alva, G., Thai, T. M. & Bunney Jr, W. E. (2005). Therapeutic effects of individualized alpha frequency transcranial magnetic stimulation (αTMS) on the negative symptoms of schizophrenia. Schizophrenia bulletin, 32(3), 556-561. 8. PROGRESS REPORT This is a new project. 9. RESEARCH DESIGN AND METHODS Biomedical IRB Application Instructions Page 7 Study location: The EEG data recordings are performed at the PrTMS Research Center (16918 Dove Canyon Rd, San Diego, CA 92127, suite 100). We note this location sits on the same floor (and directly adjacent to) Mindset (Suite 102) and the UCSD/cCare Radiation/Medical Oncology operation (Suite 103). UCSD faculty member Kevin Murphy, MD (Professor, RMAS) established Mindset Inc. and actively treats patients with rTMS in suite 102. UCSD specifically leased suite 100 to allow rTMS protocols developed by Dr. Murphy and the Mindset team, and other rTMS protocols, to be run outside of Mindset physical space. This spatial configuration was specifically approved by UCSD facilities. UCSD faculty member David Hoopes, MD (Associate Professor, RMAS) is physically located on site and will provide daily physician oversight of this protocol. The analysis of the EEG data will be performed at the Swartz Center for Computational Neuroscience (SCCN) in the Institute for Neural Computation (INC) at the University of California San Diego. High-density (up to 64-channel) EEG will be recorded before, during and after one session of rTMS at the PrTMS Research Center. We will measure healthy individuals with no prior neurological or psychiatric disorders that pass the safety screening described in section 10 and 11. Healthy controls will be recruited via IRB approved flyers by Dr. Scott Makeig at UCSD and in the greater San Diego area. Healthy individuals will participate in one of the three versions of the experimental protocol described in the following. Experimental Procedure: Each subject will participate in sessions lasting 80-120 minutes. The total time of an experiment will not exceed two hours. Before the experiment starts participant will be rescreened with the safety screening questionnaire (for safety screening questions see section 11 and safety screening questionnaire attached). If they reply yes to any of the questions of the safety screening they will be excluded from participation and the experiment will be terminated. In addition all women in childbearing age will be offered a pregnancy test during the consent procedure right before the EEG/TMS session. She will be asked to take the test before starting the experiment. The experimenter will administer the test. If the test is positive or if she declines to take the test she will be excluded from the experiment We will collect demographic information from the participant and participants will fill out the 20 items on stateanxiety of the State-trait Anxiety Inventory (Spielberger, 2010) to assess their current level of worry and anxiety. We want to assess whether some participants may be worried or anxious before the experiment if they have never experienced TMS before. After attachment of the electrodes, subjects will undergo rTMS stimulation for a total of 5 minutes and sham stimulation (no magnetic pulse is delivered to the head) for a total of 2.5 minutes with single stimulation periods (trains) lasting from 2-3 sec and 8-15 sec interval between stimulation trains. TMS is delivered while the electroencephalogram (EEG) is recorded. Healthy Subjects: To study brain activity during rTMS we will recruit 270 healthy subjects. Participants must be in good general health, without cognitive impairment, and between the ages of 18 and 60 years old. Healthy participants will be recruited by Dr. Scott Makeig through flyers at UCSD. Procedure of one rTMS/EEG treatment session A treatment session will proceed as follows: The subject will be seated in a quiet room, on a comfortable chair. Before each rTMS/EEG treatment session patients will fill out one standardized questionnaires to assess stress levels. Participants will wear ear plugs to minimize the clicking noise produced by the TMS pulses. Then we will determine the participant’s TMS stimulation intensity by measuring the Resting Motor Threshold. The Resting Motor Threshold (RMT) is measured by stimulating over the right primary motor cortex area with single TMS pulses at successively increasing levels until a muscle twitch is produced at the thumb of the left hand. More specifically the RMT will be defined as the intensity needed for eliciting motor evoked potentials of Biomedical IRB Application Instructions Page 8 at least 50 Microvolt in the electromyogram recorded at the thumb of the left hand in 50% (5/10) of single pulses delivered to the contralateral motor cortex. rTMS stimulation intensity will then be set to a certain percentage of motor threshold depending on the version of the experimental protocol (see below). Since the distance between the TMS coil and the cortex will increase by 5mm after placing the EEG cap (EEG electrodes are about 5mm high) we will calculate stimulation intensities using distance-adjusted thresholding (Stokes et al. 2005). Next an EEG cap specially designed by its manufacturer, Brain Products, GmBh, for use during TMS stimulation will be placed on the participant’s head and up to 64 electrodes will be attached to the scalp with water-based gel. Additional electrodes will be placed on the chest (Erb’s point) and on the right wrist to measure the electric activity of the heart. Next the participants will be seated on a comfortable chair where they can comfortably rest their head on a neck rest to avoid muscle tension and larger movements of the head. We will then record five minutes of resting EEG during which we ask the participants to relax and close their eyes. We will then determine the participant’s individual alpha frequency (IAF) (8-13Hz) in the EEG from the parietal cortex by applying a blind source separation method (Independent Component Analysis) to the EEG data and subsequently using spectral analysis. Next, we will place the TMS coil over electrode ‘Fz’ or over the dorsolateral prefrontal cortex (DLPFC) and will deliver rTMS stimulation. Stimulation parameters (location, intensity and frequency are specified in the variations of the experimental protocol below). After rTMS stimulation participants will perform a computerized mental rotation task (Klimesch et al, 2003) or a working memory task (Sternberg, 1969). It has been shown that rTMS stimulation over Fz at 1Hz above individual alpha frequency (as measured from parietal cortex) led to a significant improvement in mental rotation performance when compared to rTMS stimulation at 20 Hz and at 3 Hz below individual alpha frequency and sham stimulation. Sham stimulation in this paradigm consisted in rotating the coil by 90 deg to simulate the sensory effects of rTMS without interfering with cortical processes. Klimesch et al, 2003 showed that rTMS at 1 Hz above individual alpha frequency leads to an immediate increase of alpha power in the EEG over frontal cortex lasting up to 30 s after the end of the stimulation and to a more pronounced decrease of alpha during performance of the mental rotation task. The output strength of rTMS in Klimesch’s study was set to 110% of the subjects’ motor threshold. We will use modified versions of this protocol to investigate effects of rTMS stimulation parameters. We will use a computerized mental rotation task: described in the following: Stimuli are drawings of cubes. The cubes have different symbols on each side. On each trial a set of cubes are presented on a computer monitor. The target cube is visually marked and subjects have to decide which of the other five cubes matches the mentally rotated target. Participants will be instructed to perform the task as fast and accurately as possible. A single trial will begin with a fixation cross presented in the middle of a screen. After 1000 ms, the train of 24 TMS pulses will be delivered. Immediately after the last TMS pulse, the cubes will be presented. The next trial will begin 7 seconds after the subject responds. To assess working memory in Study 2 we will use the Sternberg task (Bailey, Segrave, Hoy, Maller, & Fitzgerald, 2014). Participants will be asked to remember a set of presented letters over a brief period and then indicate whether a single probe letter was present or not present in the memory set using a button press. The memory set will be presented with either five (low load) or seven (high load) simultaneous items (3000 ms), followed by a blank screen (3000 ms). The probe item will then be presented (2000 ms) and followed by a brief visual mask (130 ms). Responses will be required within this window to be deemed correct. Biomedical IRB Application Instructions Page 9 Figure 2. Example for two stimuli in the mental rotation task in Klimesch et al, 2003. The lower dice in 1a is the same as the upper but rotated in space, the lower dice in 1b is a different one than the upper dice. STUDY 1: Stimulator output intensity: Effect on alpha power and entrainment In Study 1 we aim to investigate which stimulation intensities of rTMS at individual alpha frequency (IAF) can produce observable behavioral changes and changes in cortical oscillations. Under this protocol we will therefore stimulate at 6 different intensities in a between subject design. Each participant will receive stimulation at 2 different intensities and sham stimulation. For sham stimulation we will use a specifically manufactured TMS-Sham coil by MagVenture, that mimics the sensory effect of rTMS without delivering any magnetic pulse. The coil delivers sham stimulation on side and magnetic stimulation on the other side, so that the participant will not be aware which is the sham and which are the real stimulation conditions. Participants will be divided in 3 groups of 24 subjects receiving stimulation at one of the intensities: 80%, 60% or at 40% of motor threshold. All participants will also receive stimulation at 110% of motor threshold and sham stimulation. Conditions will be randomized so participants will not be aware which is the sham condition. To mask the coil generated clicks, acoustical white noise will be played through earphones during all trials. For the purpose of verification the blindness of rTMS condition once per each block of stimulation (110% / subthreshold / Sham) participant will answer two questions: 1.) How do you think was it a real or sham stimulation? (Answers: real / sham) 2.) How certain you are ? (Answer on scale from 0 to 100: 0% = not at all; 100 = 100% certain) Table 1: Summary of STUDY 1 Group 1 Stimulation at % of motor threshold # Participants 24 2 110% 80% Sham 3 - 24 110% 60% Sham Total - 110% 40% Sham 24 Biomedical IRB Application Instructions Page 10 72 Stimulation time 24 pulses per train (2.4 seconds), 5 seconds inter train interval, 60 trains per stimulation intensity, 2.4 minutes stimulation per intensity Experiment structure Stimulation is performed in blocks per intensity, each block lasts 12 minutes. 20 minutes break in between stimulation blocks. 3 blocks total per experiment (one per stimulation intensity and sham). 5 minutes total rTMS stimulation at % of motor threshold, 2.4 minutes sham stimulation 80 minutes experiment 100 minutes total time including preparation of EEG Each participant will undergo a 100-min session consisting in total of three experimental conditions. To minimize plasticity effects in the excitability of the stimulated cortical area, each of the three experimental blocks will be separated by a break of 20 minutes. The order of presentation of the three blocks will be counterbalanced across participants. Two conditions of alpha-range rTMS, and one sham condition will be applied over electrode position Fz at strengths, with respect to the individual RMT of (1) 110%, (2) 80% or 60% or 40%, and (3) sham (0%). To keep the ‘total’ energy applied to the cortex by rTMS constant for the different intensities, a constant number (24) of pulses per train will be delivered. Per condition, 60 trains each lasting around 2.4 sec will be delivered. Stimulation will be delivered in blocks of 60 trials, stimulation intensity will be kept constant across bocks. A single trial will start with presentation of a fixation cross. After 1 sec, the first train of 24 pulses will be delivered. Immediately after the last TMS pulse, the cubes will be presented for 3 sec. The next trial will begin 4 sec after the subject responds. In the beginning of the trial, a fixation cross appears for 1 second before the TMS stimulation starts. There will be 60 trials in each block, each block will last around 12 minutes (stimulation intensity will be kept constant across blocks). After each stimulation block participants, will take a break for 20 minutes during which they can choose to watch a movie of their choice. Data analysis: We will compare stimulation at different intensities and sham. We will perform one-way ANOVAs with the factor stimulation intensity to analyze the behavioral data (dependent variables: percentage of correct responses and reaction time). To analyze the EEG data we will perform Independent Component Analysis on the EEG data to separate brain sources and non-brain (artifact) sources. ICA performs blind source separation of EEG data, based on the assumed temporal near-independence of the effective EEG sources (Makeig et al., 2002). We will assess the relationship between spectral power in the alpha band after the stimulation block to behavioral performance in the mental rotation task. We will segment the EEG data relative to train onsets to compare alpha power increase relative to rTMS train onset between stimulation intensities. We will also assess spectral power changes over the course of each stimulation block: e.g., Does alpha spectral power increase after consecutive stimulation trains? We will also assess phase locking of cortical alpha oscillations to TMS pulses. STUDY 2: Stimulator output intensity effect on GABA depression. In Study 2 we aim to investigate which stimulation intensities can produce observable behavioral changes and changes in cortical excitability and inhibition. Under this protocol, we will therefore stimulate at 6 different intensities in a between subject design. Each participant will receive stimulation at 3 different intensities. Participants will be divided in 3 groups of 24 subjects receiving stimulation at one of the intensities: 80%, 60% or at 40% of motor threshold. All participants will receive stimulation additionally at 110% of motor threshold and sham stimulation. Conditions will be randomized so participants will not be aware which is the sham condition. To mask the coil generated clicks, acoustical white noise will be played through earphones during all trials. Biomedical IRB Application Instructions Page 11 Each participant will undergo a 130-min session consisting in total of three experimental conditions. To minimize plasticity effects in the excitability of the stimulated cortical area, each of the three experimental blocks will be separated by a break of 20 min. The order of presentation of the three blocks will be counterbalanced across participants. Three 6-min conditions of alpha range rTMS over dorsolateral prefrontal cortex (DLPFC) will be applied with respect to the individual RMT: (1) 110%; (2) 80% or 60% or 40%; and (3) sham. Location of DLPFC will be approximated by positioning the coil so the center rests between the F3 and F5 electrode and the handle is rotated to a 45 angle relative to midline, producing a posterior-anterior current flow in the underlying cortex (following the methods in Rogasch et al, 2015). This position provides the most accurate estimation of left DLPFC (border of BA9 and BA46) in the absence of neuronavigational equipment (Fitzgerald et al., 2009). The coil border will be marked using a felt tipped pen to allow repositioning. Before starting the experimental conditions we will assess of baseline conditions of GABA transmission in DLPFC by delivering 50 single and 50 paired (interstimulus interval, 100 msec) TMS pulses for 3 minutes total to the left DLPFC while they sit quietly and look directly ahead with their eyes open. Both single and paired pulses will be delivered at 100% RMT and will be randomly interleaved at a rate of 0.2 Hz. To keep the ‘total’ energy applied to the cortex by rTMS constant for the different intensities a constant number of 24 pulses per train will be delivered. Per condition 40 trains each lasting around 2.5 sec will be delivered. Stimulation will be delivered in blocks of 40 trials, stimulation intensity is kept constant across bocks. One trial will proceed as follows: Participants will receive 24 pulses of rTMS at individual alpha frequency (IAF). 1 sec after the last TMS pulse, participants will then receive a single and paired (interstimulus interval. 100 msec) TMS pulses randomly interleaved at a rate of 0.2 Hz (10% jitter) to the left DLPFC while they sit quietly and look directly ahead with their eyes open. Order of single and paired pulse TMS will be randomized. One sec after the last TMS pulse the memory set of the Sternberg task is presented on a computer screen in front of the participant with either five (low load) or seven (high load) simultaneous items for 3 sec), followed by a blank screen (3 sec). The probe item will then be presented (2 sec) followed by a brief visual mask (130 ms). Responses within this window to be deemed correct. The next trial will start 4 sec after the subject responds. After each stimulation block participants will take a break for 20 minutes during which they can choose to watch a movie of their choice. Table 2: Summary of STUDY 2 Group 1 Stimulation at % of motor threshold - 2 110% 80% Sham 3 - - 110% 40% Sham # Participants 24 Stimulation time 24 pulses per train (2.5 seconds), 17 seconds inter train interval 40 trains per stimulation intensity, 2 minutes effective rTMS stimulation time per block, 3 minutes paired and single pulse stimulation at the beginning of the experiment Stimulation is performed in blocks per intensity, 40 trains per stimulation intensity, 13 minutes per block, 20 minutes break in between stimulation blocks. 3 blocks total per experiment (one per stimulation intensity and sham). Experiment structure 24 110% 60% Sham Total 24 Biomedical IRB Application Instructions Page 12 72 Total 4 min of rTMS stimulation and 2 min sham stimulation 100 minutes experiment 120 minutes total time including preparation of EEG Data analysis. We will compare stimulation at different intensities and sham. We will perform one-way ANOVAs with the factor stimulation intensity to analyze the behavioral data (dependent variables: percentage of correct responses). To analyze the EEG data we will perform Independent Component Analysis on the EEG data to separate brain sources and artefact sources. We will assess the relationship between entrainment of alpha power to the TMS pulse and spectral power in the alpha band after the stimulation block to behavioral performance in the working memory task. We will segment the EEG data relative to train onsets to compare alpha power increase relative to rTMS train onset between stimulation intensities. We will also assess spectral power changes over the course of each stimulation block i.e. does alpha spectral power increase after consecutive stimulation trains? We will also analyze TMS evoked potentials (TEPs) relative to single and paired pulse TMS to assess GABAergic Neurotransmission in the DLPFC after different intensities of rTMS. We will relate these measures to behavioral performance in the working memory task. STUDY 3: Individual alpha frequency. Study 3 will examine the optimal stimulation frequency in the alpha range. We will identify the individual alpha frequency as described in STUDY 1. In experiment A we will stimulate 20 participants at individual alpha frequency (IAF), and at IAF ±2Hz, in experiment B we will stimulate 20 participants at IAF and at IAF ±1Hz. We will measure behavioral effects of stimulation using the same mental rotation paradigm as in STUDY 1. We will measure effects on the EEG by assessing EEG spectral power in the alpha band pre- and post- stimulation and alpha phase locking to stimulation pulses. If results from experiment A and B show a significant difference between IAF and IAF±1Hz in behavioral data and pre/post stimulation EEG spectral power, we will perform the same experimental paradigm but stimulating at IAF and IAF ±0.5 Hz (experiment C and D). Stimulation intensity will be defined by using the results from STUDY 1 and 2: we will use % of motor threshold that showed a significant behavioral effect and alpha spectral power pre-, post- in the EEG and alpha phase locking to the TMS pulse relative to sham. Stimulation site will be Fz. After each stimulation block participants take a break for 20 minutes during which they can select to watch a movie of their choice. Table 3: Summary of STUDY 3 Group A B Stimulation # Participants Stimulation time Experiment structure C D IAF -1 Hz IAF IAF -0. 5 Hz IAF IAF IAF +1 Hz IAF IAF+0.5 sham sham sham sham 24 24 24 24 24 pulses per train (2.4 seconds), 5 seconds inter train interval, 40 trains per stimulation intensity, 2.4 minutes stimulation per intensity Stimulation is performed in blocks per intensity, each block lasts 12 minutes, 20 minutes break in between stimulation blocks. 3 blocks total per experiment (one per stimulation frequency and sham). Total 96 Total 5 min of rTMS stimulation and 2.4 min sham stimulation 80 minutes experiment 100 minutes total time including preparation of EEG Data analysis: We will compare stimulation at different frequencies and sham. We will perform one-way ANOVAs with the factor stimulation frequency to analyze the behavioral data (dependent variables: percentage of correct responses and reaction time). To analyze the EEG data we will perform Independent Component Analysis on the EEG data to separate brain sources and artefact sources. We will assess the relationship between spectral power in the alpha band after the stimulation block to behavioral performance in the mental rotation task. We will segment the EEG data relative to train onsets to compare alpha power increase relative to rTMS train onset between stimulation intensities. We will also assess spectral power changes over the course of each stimulation block i.e. does alpha spectral power increase after consecutive stimulation trains? We will also assess phase locking of cortical alpha oscillations to TMS pulses. Biomedical IRB Application Instructions Page 13 STUDY 4: Preferred alpha phase for stimulation. One of the mechanisms by which rhythmic TMS may exert its action on behavior could be the reproduction of a natural oscillatory signature of brain activity (that is also functionally relevant). This entrainment hypothesis posits that frequency tuned rhythmic TMS causes entrainment in direct interactions with the underlying brain oscillation. Entrainment supposes (1) the induction of a distinct entrainment signature, which emerges during rhythmic TMS and whose topography and frequency reproduce the natural oscillation of the targeted generator. Entrainment also supposes that there is (2) progressive enhancement of the target oscillation in the course of the TMS train as a result of progressive synchronization by each successive TMS pulse. Finally, entrainment should (3) depend on ongoing activity of the target generator, because it is supposedly driving existing brain oscillations, as opposed to generating new artificial rhythms. The first two points will be already addressed in STUDY 1, 2 and 3. STUDY 4 will address point 3 and will investigate the interaction of the TMS pulse with the ongoing EEG alpha activity. We will determine whether there is an optimal state or a preferred phase of the alpha cycle to the TMS pulse. Stimulation intensity will be defined according to parameters determined from results in STUDY 1 and 2, stimulation frequency will be defined according to parameters determined from results in STUDY 3. One trial of mental rotation task is performed after each train of 24 pulses – this will allow us to compare behavioral performance to alpha phase entrainment and alpha phase at the start of the pulse train. In between stimulation and sham block participants take a break for 20 minutes during which they can select to watch a movie of their choice. Table 4: Summary of STUDY 4 Group Stimulation Experiment structure # participants 1 24 pulses per train (2-3 seconds), 15 seconds inter train interval, 40 trains per Block, Two blocks, 2 minutes stimulation per Block Stimulation is performed in 3 blocks (2 stimulation blocks, 1 sham block), 20 minutes break in between sham and stimulation blocks. 3 blocks total per experiment (two for rTMS one for sham). 30 Total 4 minutes stimulation, 2 minutes sham stimulation 60 minutes experiment 80 minutes total time including preparation of EEG Data analysis: We will compare stimulation at different intensities and sham. We will perform one-way ANOVAs with the factor alpha phase at stimulation train onset to analyze the behavioral data (dependent variables: percentage of correct responses and reaction time). To analyze the EEG data we will perform Independent Component Analysis on the EEG data to separate brain sources and artefact sources. We will assess the relationship between spectral power in the alpha band after each train to behavioral performance in the mental rotation task. We will segment the EEG data relative to train onsets to compare alpha power increase relative to rTMS train onset between stimulation intensities. We will sort these segments by the phase of alpha oscillations at the onset of stimulation trains. This will allow us to look at relationships between alpha phase at onset and spectral power increase at alpha frequencies over the stimulation train. We will also assess phase locking of cortical alpha oscillations to TMS pulses. Biomedical IRB Application Instructions Page 14 Table 5: summarizing the studies under this protocol: Study Protocol Diagnosis PartiInterventio Time cipants n 1 Stimulation Healthy 72 Single 100 min Intensity Controls EEG/rTMS (effect on alpha No neurosession power) logic or mental 2 Stimulation 72 Single 120 min Intensity (effect disorder EEG/rTMS 3 4 on GABA) Stimulation Frequency Preferred Alpha Phase 96 30 session Single 100 min EEG/rTMS session Single 80 min EEG/rTMS session Inclusion Data collected No EEG, behavioral data in Neurologic mental rotation task, or mental STAI questionnaire disorders EEG, behavioral data in working memory task, STAI questionnaire EEG, behavioral data in mental rotation task, STAI questionnaire EEG, behavioral data in mental rotation task, STAI questionnaire Methods EEG methods. Electroencephalography (EEG) consists of summed electrical activities principally originating in small areas of cortical neuropile whose summed activities are recorded from the scalp. To collect EEG data, electrodes are placed on the scalp. All EEG recordings are bipolar; that is, they represent the difference in potential between an active electrode of interest, and a reference electrode that is treated as relatively electrically inactive. Similar to other electrophysiological signals, EEG signals are small (in the microvolts range) and need to be amplified by an amplifier designed to measure physiological signals. The procedure poses no risk whatsoever and is completely painless EEG recordings will proceed as follows: A stretchable cap containing 64 small electrodes (Acticap, Brain Products, Gilching, Germany) will be placed on the participant’s head. We have four sizes of cap that will be comfortable for a wide range of head sizes. We will be using very thin active electrodes specifically designed by the manufacturer for TMS/EEG applications. We will fill the EEG electrodes with transparent non-abrasive Electrolyte Gel for Active Electrodes using syringes with plastic heads to minimize impedance of electrical connection between electrode and scalp. Due to the highly conductive gel and active electrodes the required signal quality can be obtained without any abrasion. This leads to a significant reduction in preparation time to around 10-15 minutes. The gel is also water soluble and non-staining which minimizes discomfort to the participant. The cap and gel are removed at the end of the experiment and the skin is cleaned with water. A very small amount of gel may remain in the hair, but will wash out easily with shampoo. EEG will be recorded with a Brain Amp amplifier (Brain Amp, Brain Products, Gilching, Germany). This amplifier allows to digitize the EEG with a very high sampling rate, and accordingly reduce the TMS artefact in the EEG to below 1ms. All EEG recordings are performed during rTMS therapeutic sessions. TMS methods. TMS is applied through a magnetic stimulator. The stimulator consists of a set of electrical capacitors which can store and rapidly discharge electricity into a coil of electrical wire that is encased in shielded plastic. We will use a figure-8 shaped coil, composed of two rings, each 90 mm in diameter. The weight of the coil is approximately 1 kg. The volunteer sits upright in a chair and the coil is placed against the head in Biomedical IRB Application Instructions Page 15 various positions. Sometimes the coil is held in position using a tripod device. On each pulse (triggering) of the TMS machine, a brief (< 1 ms) magnetic field is produced by passing a powerful and rapidly changing current through the wire coil. According to Faraday’s law, the electrical current flows through the coil, and a magnetic field is generated that penetrates skin and skull and induces a second electrical flow of current in the brain. There is also a degree of movement of the coil within its plastic casing which produces a “click” sound. The induced current in the brain very briefly alters neural activity (for about 1/10 of a second, or less) in brain areas lying directly beneath the coil, by causing discharge of (perpendicular) pyramidal neurons at a depth of 1.5 to 2.0 cm below the scalp. When placed over the motor cortex, TMS leads to brief activation of descending motor pathways, and this produces a detectable muscle twitch in the wrist for example. This allows to measure the resting motor threshold to determine the strength of stimulation for rTMS. We plan to apply TMS in several ways: i) repetitive TMS at 10 Hz over the Fz electrode or over the left Dorsolateral Prefrontal Cortex (between F3 and F5 electrode) using the Figure-of-Eight coil. Trains of 2.5 sec (24 pulses each) with 15 sec inter train interval. ii) Single pulse TMS over the Fz electrode or over the left Dorsolateral Prefrontal Cortex (between F3 and F5 electrode) using the Figure-of-Eight coil. iii) Paired pulse TMS over the Fz electrode or over the left Dorsolateral Prefrontal Cortex (between F3 and F5 electrode) using the Figure-of-Eight coil; with the inter-pulse interval of 100ms. Stimulation strength will be set according to study protocol above and adjusted relative to the height of the EEG electrodes. The distance between the TMS coil and the cortex will increase by 5mm after placing the EEG cap (EEG electrodes are about 5mm high). We will therefore calculate stimulation intensities using distanceadjusted thresholding (Stokes et al. 2005). For every millimeter from the stimulating coil, an additional 3% of TMS output is required to induce an equivalent level of brain stimulation at the motor cortex. rTMS stimulation frequencies will be applied at individual participant’s alpha frequencies as determined from EEG. TMS Investigational Device The TMS investigational device used in this study is the MagVenture MagPro R30 magnetic stimulator paired with the MagVenture Cool-B65 Active/Placebo Coil (magnetic paddle). We believe that the device meets the Federal Drug Administration (FDA) definition of nonsignificant risk and thus quality for inclusion in our protocol without submission of an FDA investigational device exemption (IDE). Magnetic stimulation is generally regarded as a non- significant risk procedure in normal populations. The FDA defines significant risk (SR) and non-significant risk (NSR) devices as follows (https://www.fda.gov/downloads/regulatoryinformation/guidances/ucm126418.pdf): A. What is a Significant Risk Device Study? Under 21 CFR 812.3(m), an SR device means an investigational device that: • Is intended as an implant and presents a potential for serious risk to the health, safety, or welfare of a subject; • Is purported or represented to be for use supporting or sustaining human life and presents a potential for serious risk to the health, safety, or welfare of a subject; • Is for a use of substantial importance in diagnosing, curing, mitigating, or treating disease, or otherwise preventing impairment of human health and presents a potential for serious risk to the health, safety, or welfare of a subject; or • Otherwise presents a potential for serious risk to the health, safety, or welfare of a subject. B. What is a Nonsignificant Risk Device Study? An NSR device study is one that does not meet the definition for an SR device study. Biomedical IRB Application Instructions Page 16 The MagPro R30/Cool-B65 is not implanted, does not support or sustain human life, and does not present the potential for serious risk to the health, safety, or welfare of a subject. We offer the following as justification. Concerning the MagVenture MagPro magnetic stimulator and treatment coil used in this study, we note that the MagPro stimulator is FDA approved for the treatment of major depressive disorder in adult patients who have failed to receive satisfactory improvement from prior antidepressant medications in the current episode (501(k) number K150641) – (see 501(k) summary attached to the IRB). This is a class II medical device (https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=882.5805). According to information by the manufacturer no Investigational Device Exemption from the FDA exists or has been requested for research in healthy subjects involving the MagVita TMS Therapy System. Magnetic stimulation is generally regarded as a non-significant risk procedure in normal populations. We note that our experimental TMS protocol (detailed in section 9 under Experimental procedure) is similar to other research studies on healthy participants (see section 9 under Safe parameters for rTMS in physiological or cognitive brain research). The parameters we are using are in the safe range according to published guidelines (see Rossi et al, 2009). The risk to healthy participants in our study is not increased over an FDAapproved treatment, and our study population does not represent an increased risk. We therefore do not believe that the MagVenture MagPro magnetic stimulator and treatment coil presents a potential for serious risk to the health, safety, or welfare of subjects and is best classified as NSR device. The following examples are also instructive. We note that in the FDA list of example nonsignificant risk devices, the following two entries are listed: • Functional Non-Invasive Electrical Neuromuscular Stimulators • Magnetic Resonance Imaging (MRI) Devices within FDA specified parameters We note that the MagProR30 (our stimulator device) is also already FDA approved as a functional Non-Invasive Electrical Neuromuscular Stimulator (510(k) K091940). We also note that MRI devices are considered nonsignificant risk as long as the main static magnetic field strength is less than or equal to 8 Tesla for patients > 1 month of age (https://www.fda.gov/downloads/medicaldevices/deviceregulationandguidance/guidancedocuments/ucm0726 88.pdf). The maximum field strength for the Cool B-65 Active/Placebo Coil driven by the MagPro R30 stimulator, as stated by the manufacturer, is 2.7 Tesla. Note that this value 2.7T is far below 8T, and is similar to the field strength used in research on healthy participants involving MRI (~3T). The TMS device used and the procedures therefore do not represent a greater risk to participants than standard MRI. In conclusion, we believe that the MagVenture MagPro R30/Cool-B65 Active/Placebo Coil (magnetic paddle) does not present a potential for serious risk to the health, safety, or welfare of our subjects. In fact the procedure does not represent increased risk for the participants compared to standard MRI procedures. We believe therefore that this device is best classified as nonsignificant risk (NSR). We do not believe it requires submission of FDA IDE. Neuronavigation system. We will also be using a neuronavigation system, which includes a camera and a tracking tool which tracks the position of the coil relative to the head of the participant during the experiment. The position of the participant’s head and the TMS coil will be tracked using a camera and three plastic-coated metallic markers placed on the participant’s face. The markers will be attached using self-adhesive stickers, and are easy to remove after the experiment. The three markers (small, light plastic-coated metal discs) will be placed separately on the nose and below each eye. Note that the camera is not recording any video nor pictures; it is merely recording and tracking the position of the markers on the head of the participant and on the TMS coil. We are using the ANT neuro neuronavigation system, a commercial hardware and software solution for improving TMS coil positioning. This will allow a more precise positioning of the TMS coil. We will begin with standard head models, Biomedical IRB Application Instructions Page 17 which will then be adjusted to the experimental participant’s measured scalp dimensions and cortical locations previously identified by other EEG or fMRI studies (we will not collect individual MRI scans in this protocol). Individual participant EEG electrode placement will be identified using the get_chanlocs system, an EEGLAB plug-in for Matlab developed at UCSD by scientists at the Institute for Neural Computation. get_chanlocs will digitize the positions of electrodes on a subject’s head recorded from a 3-D camera, and will enter them into the EEG.chanlocs data structure for use in the ANT neuro neuronavigation system. While we will not store subjects’ images since facial features will be blurred after recording, the initial acquiring of 3-D images will record subject’s heads. Therefore, all subjects who are enrolled in the study will also be required to sign UCSD’s informed consent form regarding the release of head images. Locations of stimulation will remain roughly the same as described in STUDY 1 and STUDY 3 with a fine tuning of the coil position. The procedure poses no risk whatsoever and is completely painless. Information on the TMS system. rTMS will be administered using a MagPro R30 (a magnetic stimulator) manufactured by MagVenture paired with the MagVenture Cool-B65 Active/Placebo Coil (magnetic paddle). MagPro R30 is a versatile magnetic stimulator. Specific application of the device can be arranged by selection of numerous coil configurations, rate (single pulse up to 30/sec), and output intensity (up to 2.7 tesla) depending on the triggering program and type of coils. This device is currently used for the examination of the physiology of the motor pathways in the central and peripheral nervous system in clinical neurological practice, and research in transcranial magnetic stimulation and rTMS. The Cool-B65 Active/Placebo Coil is able to function as both an active and placebo coil and is specifically designed for use in double-blinded studied. A technician trained in the TMS delivery and appropriate response to medical emergencies, including syncope and seizure, will be physically present with the participant throughout the duration of TMS delivery. Delivery of Sham rTMS. We will use the MagVenture Cool-B65 Active/Placebo Coil for both active and sham rTMS. The Cool-B65 Coil is a symmetrical coil with no indication of active versus placebo sides. In one orientation the Cool-B65 treats actives rTMS and in the other orientation (flipped over, 180 degrees) the CoolB65 delivers sham rTMS. There is a built-in orientation switch which determines which side of the coil to place towards the patient and is communicated to the TMS technician with a screen prompt using the rTMS research software. The system also includes adjustable output for current stimulation of the patent’s skin synchronously with the magnetic stimulation pulses to account for the scalp sensation/twitch noted by some participants. The procedure for Sham rTMS will be identical to that of active rTMS. All parties, including the participant, rTMS technician and principal investigator, will be blinded to treatment arm. Safe parameters for rTMS in physiological or cognitive brain research. Rossi et al, 2009 conducted a survey of the studies that have used TMS trains to interact with task performance in cognitive science from 1999 to December 2008. In these protocols, short trains of a few hundreds of ms to several seconds are applied online to task performance on the trial level (usually aligned to trial onset). These protocols have been widely employed in healthy volunteers without side-effects, following the publication of the previous 1998 safety guidelines (Wassermannn, 1998) and after screening via the safety questionnaire to eliminate contraindications (see Section 11). This has resulted in a large collection of empirical data for TMS applications beyond singlepulse, double-pulse and 1-Hz TMS in psychology and cognitive sciences (see Rossi et al, 2009, Supplementary Material). Over the last 10 years, 4-Hz to 25-Hz trains have been tailored to cover usually 0.1–3 s and exceptionally up to 30 s of task performance. More than 50 studies used 10 Hz, more than 20 have employed 20–25 Hz and more than 10 studies used 4–9 Hz. Parameters to consider for designing experiments are the duration of the TMS-train, the stimulation rate (in Hz), the inter-train interval and the number of trials within the experiment. For safety aspects, the combination of parameters is important, with short train durations and long inter-train intervals carrying less risk. To avoid possible side-effects also in the future and to remain within safe margins, future studies using the online interaction protocols should design their parameters to fall within the Biomedical IRB Application Instructions Page 18 range previously used (see Table 5, and Rossi et al, 2009). The parameters we are using in our studies remain within the range of parameters previously used in rTMS research studies reported in the literature (see Table 6 and 7). Table 6: rTMS stimulation parameters used in cognitive brain research studies Authors Rushwort h et al. Klimesch et al Turatto et al. Sack et al. Chambers et al. Feredoes et al. Hamidi et al. Probric et al. Schenklu hn et al. Hamidi et al. Tremblay et al. Frequen cy (Hz) Number of pulses Train duration Inter-train interval [s] Intensity Total real pulses n of trials with tms 10 Hz 10 Hz + 1 Hz 6 500ms ≥3.47 100% MT 1260 210 24 3000ms 15s 110% 1728 72 Parietal Frontal, Parietal 8 3s+RT 160 frontal 0-5.7s 100%MT 80% stim out 1280 3 +600 700ms 200ms +600s 480+1200 160 Parietal 2006 10 Hz 10 Hz + 1 Hz 10 Hz (Exp2) 4 300ms 2.5s+RT 120%MT 1920 480 2007 10 Hz 30 3000ms 10s 110%MT 2160 72 2008 10 Hz 30 3000ms 12.5s 110%MT 2160 72 2008 10 Hz 6 500 ms 5s 2008 10 Hz 5 400ms 1.21.9s+RT 110%MT 40% or 120% of MT 2304 7200 (2400*3day s) 384 1440 (480*3days ) parietal frontal, temporal frontal, parietal frontal, temporal, vertex 2009 10 Hz 30 3000ms 12-13s 10 Hz 5 400ms 6.9±1.5s 2160 1200 over 3 hrs 72 2009 110%MT 110%MT (rMT) Parietal frontal, parietal 240 frontal Year 2001 2003 2004 2005 stim lobe Table 7: rTMS stimulation parameters used in our studies STUDY1 Frequenc y (Hz) ~10 Hz Number of pulses 24 Train duration 3000ms Inter-train interval [s] 15s STUDY2 ~10 Hz 24 3000ms 17s 40%110%MT STUDY3 ~10 Hz 24 3000ms 15s STUDY4 ~10 Hz 24 3000ms 15s 80%MT or below 80%MT or below Intensity 40%110%MT Total pulses 2880 real n of trials with tms 120 stim lobe frontal 1920 rTMS + 390 paired pulses 2880 80 frontal 120 frontal 1920 80 frontal The safety of rTMS in clinical settings: rTMS is a noninvasive method to cause depolarization or hyperpolarization in the neurons of the brain. It uses electromagnetic induction to induce weak electric currents using a rapidly changing magnetic field. These weak electrical currents may cause activity in specific or general parts of the brain. There is typically no or very little discomfort with rTMS. A variety of rTMS protocols have been tested as a treatment tool for various neurological and psychiatric disorders including migraines, strokes, Parkinson’s disease, dystonia, tinnitus, depression and auditory hallucinations. In no disease site, or TMS application, have adverse events been found to limit the use of TMS clinically. Biomedical IRB Application Instructions Page 19 The disease with the most published rTMS data is depression. The Clinical TMS Society published a consensus review and treatment recommendation for TMS in depression in 2016 (Perera et al, 2016). The risk of TMS are minimal, rare, and limited to headache, scalp discomfort, syncope and seizures. Perhaps the most concerning risk of TMS is seizure. The seizure risk is reported to be 1.4% (crude estimate) in known epileptic patients and <1% in patients without a seizure history (Rossi et al, 2009). The consensus guideline describes the incidence of seizure with TMS as small and slightly lower than the seizure risk reported for the use of current antidepressant medications (Voigt et al, 2017; Breden Crouse, 2012). The overall seizure risk is estimated to be less than 1 in 1000 patient exposures and less than 1 in 30,000 treatment sessions. There is no need to take any special seizure precautions with TMS outside of staff training regarding the proper first responder actions for seizure. The following statement appears in the Clinical TMS Society consensus document (Perera et al, 2016): It is the consensus of the cTMSs that IV access, cardiac defibrillators, suction, and oxygen are NOT necessary for the safe administration of TMS in an outpatient TMS office. In placebo-controlled randomized trials of TMS for depression, TMS has been very well tolerated. In one clinical trial, the dropout rate for adverse events was 4.5% and adverse events were generally mild and limited to scalp discomfort (Breden Crouse, 2012). In another placebo controlled randomized trial, there were no statistically significant differences in adverse events between the active treatment arm and the sham arm for any parameter including headache, scalp discomfort, insomnia, worsening depression or anxiety, gastrointestinal symptoms, fatigue, muscle aches, vertigo, skin pain, facial twitching or other (Wajdik et al, 2010). In a third placebo controlled randomized trial of TMS in depression, the only adverse event found to differ between TMS and placebo was application site (scalp) pain (5% vs 0%; p=0.02) with no statistical difference seen in all other parameters including headache, muscle twitching, back pain, anxiety, and insomnia. This trial did report a single case of device-related serious adverse event (seizure). This patient had a two minutes generalized seizure during TMS, was managed with several hours observation in the Emergency Department, released without medical intervention, and had no reported sequelae as a result of the event. This event was reported to the FDA. In addition, we note that in the FDA approval for TMS (a class 2 device) for the treatment of major depressive disorder (https://www.fda.gov/RegulatoryInformation/Guidances/ucm265269.htm) specific guidance is given regarding the risk of seizure. The potential biologic effect of TMS on the CNS, including the risk of seizure, is related to the following variables: Table 8: Potential biologic effect of TMS on the CNS Variable Definition Frequency Intensity Train Duration The number of magnetic stimulations (cycles) per second The strength of the magnetic stimulation as measured by the percent of the motor threshold (i.e. intensity needed to provoke a hand twitch with stimulation of the motor cortex) The duration of magnetic stimulation before a planned break. Biologic Effect increases with Increasing frequency Increasing intensity Increasing train duration As part of FDA approval, data are presented on the maximum safe train duration limits (in seconds) for avoiding seizure given different intensities and frequencies. Please note that the total number of pulses is not listed as Biomedical IRB Application Instructions Page 20 one of the factors increasing the risk of seizures. In fact, clinical trials for depression treatment with rTMS have used the following parameters in a total of 491 patients: 10Hz stimulation frequency, train duration 4sec, intertrain interval 26sec, 120% of motor threshold, 3000 pulses total per session per day (O’Reardon et al., 2007, George et al, 2010, Perera et al, 2016). This data comes from published articles (Rossi et al, 2009, Wassermann et al, 1998, https://www.fda.gov/RegulatoryInformation/Guidances/ucm265269.htm) and is reproduced in full in Table 9: Table 9: Maximum Safe Train Duration (seconds) Limits for Avoiding Seizure Maximum Safe Train Duration (seconds) Limits for Avoiding Seizure Freq (Hz) 1 5 Intensity (% of Motor Threshold) 80-100 100 110 120 >1800 >1800 >1800 360 >10 >10 >10 >10 130 >50 >10 140 >50 7.6 150 >50 5.2 160 >50 3.6 170 27 2.6 180 11 2.4 190 11 1.6 200 8 1.4 210 7 1.6 220 6 1.2 10 >5 >5 >5 4.2 2.9 1.3 0.8 0.9 0.8 0.5 0.6 0.4 0.3 0.3 20 2.05 2.05 1.6 1.0 0.55 0.35 0.25 0.25 0.15 0.2 0.25 0.2 0.1 0.1 25 1.28 1.28 0.84 0.4 0.24 0.2 0.24 0.2 0.12 0.08 0.12 0.12 0.08 0.08 Table 10: Safety parameters for rTMS (from TMS consensus paper Rossi et al, 2009) In the current protocol TMS parameters are determined based on the individual alpha peak over parietal cortex in the EEG. Table 10 shows the possible range of rTMS parameters for this protocol: Table 11: Possible range of rTMS parameters for this protocol compared to Rossi et al 2009 safety range Frequency Intensity Train Pulses Inter-train (% resting Duration per train interval motor threshold, RMT) Our protocol 8-13 Hz 40-110% 2.5-3 24 5 sec seconds Safe rTMS parameters 10 Hz 110% Max 5 Max 50 Min 5 sec (according to Rossi et al, sec 2009) Biomedical IRB Application Instructions Page 21 Note that the rTMS parameters for this protocol all remain well inside the safety recommendations from the Rossi et al, 2009 consensus paper on the safety of rTMS. When comparing the range of stimulation parameters in the current protocol, to Rossi et al 2009 safety reccomendations (see Table 11), the maximum safe train duration according to Rossi et al 2009 is 5 seconds and 50 pulses. The trains used in this protocol last 2-3 seconds and contain 24 pulses are therefore in a safe range. Also our inter-train interval remains inside the safety guidelines. Minimum safe inter-train interval is 5 sec, our protocol uses 5 sec. In summary when following the safety guidelines and exclusion criteria, rTMS represents a safe procedure with very minimal side effects limited to uncommon scalp pain, headache, syncope and very rare seizure EMG methods: EMG stands for electromyography. It is the study of muscle electrical signals. Electrodes are placed on the skin overlying the muscle. Similar to other electrophysiological signals, EMG signals are small and need to be amplified by an amplifier designed to measure physiological signals. The purpose of EMG in the current proposal is to measure the amplitude of the Motor Evoked Potential produced by TMS over the contralateral motor cortex, to determine the resting motor threshold for TMS stimulation. When a TMS pulse is delivered over motor cortex it causes a depolarization of pyramidal neurons leading to action potentials which become a volley of electrical activity down the corticospinal tracts and into the muscles of the hand (for example). The degree of excitability of the corticospinal tract can be indexed by the size of the resulting change in MEP amplitude recorded from the muscles. ECG methods: The electrocardiogram (ECG) is recording the electrical activity of the heart. Three electrodes are placed on the skin over the chest of the participant. Similar to other electrophysiological signals, ECG signals are small and need to be amplified by an amplifier designed to measure physiological signals. The purpose of ECG in the current proposal is to explore possible relationships between changes in heart rate and cortical oscillatory activity during rTMS. It has been shown in the past that heart rate is related to oscillatory activity in the EEG (Pfurtscheller et al, 2013). 10. HUMAN SUBJECTS We anticipate recruiting 270 participants in total. We will recruit women and men in roughly equal proportions, and we will recruit and study all eligible individuals without regard to race or ethnicity. We will exclude participants that do not have sufficient fluency in English. To clarify, minorities of all races, ethnical backgrounds, religion, gender, sexual orientation etc. are included in the study, as long as they are fluent in English and able to understand screening questions, consent and instructions during the experiment and understand the risks and procedures associated with this study. The provision to be fluent in English is solely chosen as a measure to ensure the safety of all participants. Before participation (during recruitment and again during the consent procedure right before the experiment) participants must complete (and pass) a safety-screening questionnaire. Participants will be excluded from participation of this experiment if they: - have a history of epilepsy, or a family history of epilepsy - have an implant such as a cardiac pacemaker or a cochlear implant or any metal inside your head (standard tooth fillings are fine) - are pregnant (risks or safety of rTMS to either the mother or unborn fetus have not been established) - if you have a neurologic condition such as dementia or movement disorders or have ever had a traumatic brain injury such as a concussion - suffer from frequent or severe headaches - have previously experienced claustrophobia (you may be uncomfortable with being maintained in close quarters (i.e. within an experiment requiring them to keep their heads still)) - have experienced fainting from time to time - are taking any psychiatric or neuroactive medications such as antipsychotics, mood stabilizers or Biomedical IRB Application Instructions Page 22 - 11. anticonvulsants have Diabetes mellitus, a history of stroke or if you currently use any sleep medication. (These conditions may cause an impaired ability to sense heat/pain and may pose an increased risk of thermal injury during rTMS.) are not sufficiently fluent in English as to understand the screening questions, consent and instructions during the experiment and understand the risks and procedures associated with this study. Minorities are explicitly included in the study and the provision to be fluent in English is solely chosen as a measure to ensure the safety of all participants. RECRUITMENT AND PROCEDURES PREPARATORY TO RESEARCH Inclusion and Exclusion Criteria All participants in the study will be healthy normal individuals who are between 18 and 60 years of age with sufficient fluency in English to understand the risks and procedures associated with this study. Based on the recommendations of the International Workshop on the Safety of Repetitive Transcranial Magnetic Stimulation (reported in Wassermann, 1998), see attached, we will exclude subjects on any of the following grounds (and see attached safety screening questionnaire) including the questions below. An initial safety screening will occur online before personal contact with the participant. Please note that if patients are deemed eligible participants per the online safety screening, we will conduct in-person screening visits which will include a second, physical safety screening as well as written informed consent to participate in the basic research study. Recruitment Volunteers will be recruited by Dr. Scott Makeig via IRB approved advertisements placed around UCSD campus and other clinics in San Diego. (See attached flyer for approval). All participants will be screened and recruited in conformance with the UCSD Human Subjects Committee Guidelines. The IRB-approved recruitment fliers posted around UCSD campus will provide a link (tmsscreener.ucsd.edu) which potential subjects may use to access the online safety screening questionnaire. Initial safety screening will occur online prior to any personal contact with the potential participant. The online consent to participate in the safety screening questionnaire will be hosted on the secure server of the Swartz Center for Computational Neuroscience, UC San Diego. The provided link points directly to a landing page which provides a description of the study and an explanation of the online safety screening and subsequent in-person informed consent process. If patients agree to take part in the initial online safety screening, they will be asked to complete an online survey/questionnaire. This survey/questionnaire will ask about some of their medical history (pertinent to safety guidelines recommended for TMS). If potential subjects do not meet screening criteria, their data will be discarded. If, after submitting their answers, subjects meet screening criteria, the webpage will ask for their name and contact information in order for our team to reach out for an in-office screening with the subject. The safety screening questionnaire contains the following questions: ● ● Have you ever had an adverse reaction to TMS? Excludes subjects who have had an adverse reaction Do you experience claustrophobia? Excludes subjects who are uncomfortable with being maintained in close quarters (i.e. within an experiment requiring them to keep their heads still). Biomedical IRB Application Instructions Page 23 ● ● ● ● ● ● • ● ● ● ● ● ● ● • Have you had a seizure? There is a very slight risk that TMS can induce a seizure – and this will be more likely in those who are predisposed towards seizures Have you had a stroke? There is a very slight risk that TMS can induce a seizure – and this may be more likely in people who have had a stroke Do you, or does anyone in your family, have epilepsy? There is a very slight risk that TMS can induce a seizure – and this may be more likely in people who have had a stroke Have you had a serious head injury (include neurosurgery)? There is a very slight risk that TMS can induce a seizure – and this may be more likely in people who have had a stroke Have you ever had any other brain-related condition? There is a very slight risk that TMS can induce a seizure – and this may be more likely in people who have had a stroke Have you ever had any illness that cause brain injury? There is a very slight risk that TMS can induce a seizure – and this may be more likely in people who have had a stroke Are you taking any psychiatric or neuroactive medications such as antipsychotics, mood stabilizers or anticonvulsants? There is a very slight risk that TMS can induce a seizure – and this may be more likely in people taking certain kinds of drugs Do you have any metal in your head (outside the mouth) such as shrapnel, surgical clips, or fragments from welding or metalwork? TMS induces small electric currents, so metal anywhere in the head is a contraindication. Standard tooth fillings are fine. Do you have any implanted devices such as cardiac pacemakers, aneurysm clips, cochlear implants, medical pumps, deep brain stimulators, or intracardiac lines? TMS induces small electric currents, so metal anywhere in the head is a contraindication. Standard tooth fillings are fine. Do you suffer from frequent or severe headaches? Keeping the head upright for long periods can cause muscle fatigue that might cause headaches (see below in Risks). Better to exclude people who have headaches. Are you pregnant? There is no known risk of TMS to either the mother or unborn foetus; however, the safety information of the of MagVita TMS Therapy system states that its safety and effectiveness has not been established in persons who are pregnant or nursing, therefore we will exclude this population. Do you hold a heavy goods vehicle driving license or bus license? TMS have not been shown to have long-term side-effects, but just in case, it’s better to exclude this group. Are you someone who faints from time to time? Some subjects could experience fainting/syncope during the TMS procedure, and this question is designed to exclude them. Have you ever lost consciousness because of feeling trapped? Some subjects could experience fainting/syncope during the TMS procedure, and this question is designed to exclude them. Did you have adequate sleep last night? Being well rested reduces chances of syncope. Biomedical IRB Application Instructions Page 24 • Do you have Diabetes mellitus, a history of stroke or do you currently use any sleep medication? These conditions may cause an impaired ability to sense heat/pain and may pose an increased risk of thermal injury during rTMS. If patients answer “yes” to any of the points on the safety screening questionnaire, they will be directed to a page which thanks them for participating in the safety screening and informs them they are not eligible for the study. The system will not prompt them for any additional information and their answers will be deleted. If patients answer “no” to all points on the safety screening questionnaire, they will be directed to a page which prompts them to provide their email address so that a research coordinator may reach out to them for an inperson screening appointment. A study coordinator will then contact potential subjects by email to set up an appointment to come to the research center. The coordinators will also provide potential participants with a copy of the study’s informed consent document for their review before the appointment. At the in-person screening appointment, patients will undergo the written informed consent process before performing any research procedures for this study. After providing written informed consent, subjects will also repeat the safety screening questionnaire physically. Thus, safety screening will be administered and documented twice for all participants, and patients will undergo documented informed consent before any research procedures begin. Patients must provide consent to participate in the online screening process, which may include documentation of their contact information. No personal data will be collected before this consent is provided, and only participants who pass the safety screening will be prompted to provide their contact information. 12. INFORMED CONSENT Informed consent to participate in the online screening is obtained electronically on the UCSD server-stored online platform before participants provide any information on the safety screening questionnaire. Participants will click on ‘yes’ on the safety screening consent page if they wish to participate in the screening and potentially be contacted by study staff. We ask to waive the documented written consent for online screening and we provide justification according to item a) on the research plan instructions: “the only record linking the subject and the research at this stage would be the consent document and the principal risk would be potential harm resulting from a breach of confidentiality.” We ask to waive the documented written consent for online screening to avoid collection of participants’ personal data before they answer any of the safety screening questions. Single responses on the safety screening sheet will not be saved on the platform, only whether the participant passed the screening. During the online screening process, no personal data will be collected before participants consent to participate in the online screening process. Participants only provide their email in the case that they pass the safety screening. This way, we will avoid linking participant’s personal information to potentially sensitive answers they may provide via the safety screening questions. Written and signed informed (documented) consent is then obtained in-person when subjects come to the research center to participate in the study. Documented informed consent will be obtained before subjects participate in any research procedures for this study. Written signed consent is obtained by a research assistant/experimenter, (see section 21, PRIVILEGES/CERTIFICATIONS/ LICENSES AND ROLES OF RESEARCH TEAM for named individuals – additional personnel will be hired by UCSD in the next month and subsequently included in this protocol) who explains the nature and purpose of the study to the subject. Informed consent will occur in a dedicated separate screening room at the UCSD PrTMS Research Center, immediately adjacent to the room where the EEG/TMS experiment will take place. Informed consent will take Biomedical IRB Application Instructions Page 25 place in an interview setting to ensure that potential participants understand each part of the study, with special attention paid to potential risk factors, and with the TMS equipment in view. During the in-person screening visit, participants are also asked to fill out the TMS safety screening questionnaire again. If they answer “yes” to any of the questions, they will be excluded from the study and recorded as screen failures. All subjects are told that the benefit of participating in the study is that we will gain scientifically relevant information, but there is no other direct benefit to them from participating in the study. Special care will be taken during the consenting process when briefing subjects regarding the possibly uncomfortable effects of TMS (see below) in terms of neck stiffness (due to posture), the clicking made by the coil (requiring ear plugs) and the mild tapping feeling against the head and possibility of headaches (due to muscle tension). It will be emphasized that the participant should keep the experimenter apprised as to how he or she is feeling at regular intervals. All subjects are told that their participation is completely voluntary and that they may refuse to participate if they wish, with no negative consequences. All subjects are told that they may withdraw from the study at any time and for any reason (without needing to provide the reason) without any negative consequences. No subjects are coerced in any way to agree to participate or to continue to participate in the study. In addition to receiving a copy of the signed documented informed consent, all enrolled subjects receive a copy of the "Experimental Subject's Bill of Rights." 13. ALTERNATIVES TO STUDY PARTICIPATION This is not a therapeutic study. The alternative to participation is to not participate in the study. 14. POTENTIAL RISKS Two potential concerns with TMS arise from discomfort during the stimulation, and risk of seizure induction. There are no known risks associated with skin surface EEG, EMG or ECG recording, as using current highimpedance amplifiers greatly reduces the need to first clean the skin abrasively. TMS: a. Discomfort of TMS: For many scalp locations, TMS feels only like a sudden mild tap on the scalp accompanied by a clicking sound. For other scalp locations, and depending on the intensity of stimulation, TMS can cause brief activation of face muscles, or muscles in the hand or arm. These brief twitches are not painful, but can feel odd or disquieting when first experienced. We will closely monitor subjects' comfort and will request them to tell us if the stimulation ever feels painful. If this occurs, we will immediately decrease TMS intensity to a level that is not painful. Participants will also be offered acetaminophen (Tylenol) before the EEG/TMS session to alleviate possible experience of headache during TMS. • Approximately 3 to 10% of people undergoing TMS experience headaches, which are believed to be due to excessive muscle tension. In the case of a headache, participants will be advised to take their normal measures to resolve it (such as returning home and resting). The experimental procedure will be immediately terminated whenever anyone reports experiencing a headache. The headaches are not recurring and subside following termination of the procedures. Biomedical IRB Application Instructions Page 26 • Approximately 1% of people undergoing TMS experience neck stiffness and neck pain. This is believed to be due to the straight posture of the head and neck during the application of TMS. If the subject wishes it, the experimental procedure will be immediately terminated. There are no reports of any such effects recurring. • TMS can produce a clicking noise when the current passes through the coil, depending on the intensity of stimulation. To prevent any adverse effect on hearing all experimental participants will wear either earplugs or headphones (through which the auditory tones can also be presented). Animal and human studies have demonstrated that earplugs can effectively prevent the risk of hearing disturbances or discomfort due to TMS. The experimenters will also be provided with ear protection devices to minimize any discomfort they might experience from the clicks. The ear protection devices reduce the intensity level of the click to approximately 80 dB. • In cases where the TMS coil is positioned over the cerebellum, temporary nausea has been reported in up to 25% of the population. This experience of nausea does not last for more than a few minutes after stimulation. No other side effects, either short-term or long-term, have been reported after experiences TMS-induced nausea. The current line of research does not propose stimulation over the cerebellum. • Some subjects might find TMS a stressful experience, especially in the first few minutes (while they get used to it). Such stress is likely to be exacerbated by not having slept well, or having missed breakfast. b. Contraindications of TMS • Some subjects who are uncomfortable with being maintained in close quarters (i.e. within an experiment requiring them to keep their heads still) may experience the feeling to be trapped and could experience fainting/syncope during the TMS procedure. • TMS induces small electric currents, so metal anywhere in the head such as shrapnel, surgical clips or implanted devices such as cardiac pacemakers, aneurysm clips, cochlear implants, medical pumps, deep brain stimulators, or intracardiac lines are a contraindication. If a TMS coil is discharged close to the implanted wires connecting the electrodes to the implanted device, potentially significant voltages and currents could be induced, which could cause unintended neural stimulation and may present a safety risk. • There is no known risk of TMS to pregnant women (either the mother or unborn foetus); however, the safety information of the of MagVita TMS Therapy system states that its safety and effectiveness has not been established in persons who are pregnant or nursing. • There is a small risk of seizure induction by rTMS (see next point c. Risk of seizure induction or syncope). This will be more likely in people who have a neurological condition such as epilepsy, dementia, motor disorders, head injury or brain injury. • People who are taking any psychiatric or neuroactive medications such as antipsychotics, mood stabilizers or anticonvulsants may have an increased risk for seizure induction. • There is a very small risk of thermal injury during rTMS in the case participants have a condition of Diabetes mellitus, a history of stroke or if participants currently use any sleep medication. These conditions may cause an impaired ability to sense heat/pain and may pose an increased risk of thermal injury during rTMS. Biomedical IRB Application Instructions Page 27 c. Risk of seizure induction or syncope: Perhaps the most concerning risk of TMS is seizure. An international workshop on the safety of TMS held at the National Institutes of Health in June 1996 concluded that the risks of single-pulse, paired-pulse, and slow repetitive TMS (<= 1 Hz) are minimal for populations without certain predisposing conditions (such as family history of epilepsy and see other Exclusion criteria above), provided that appropriate safety guidelines and precautions are followed (Wassermann, 1998) . The workshop discussed risk factors associated with the two proposed procedures (single pulse and double pulse TMS), as well as low frequency repetitive TMS, and found them to be identical. Some labs report syncope (fainting) in some subjects, usually those who are tired/weak/sick, or find the TMS procedure stressful. The workshop report defines the critical safety factor seizure induction as related to the rate of stimulation, identifying additional risks with stimulation rates between 3 and 25 Hz. A recent consensus conference (Rossi et al, 2008), updated the previous safety guidelines for the application of transcranial magnetic stimulation (TMS) in research and clinical settings.). The consensus guideline describes the incidence of seizure with TMS as small and slightly lower than the seizure risk reported for the use of current antidepressant medications (Voigt et al, 2017; Breden Crouse, 2012). The overall seizure risk is estimated to be less than 1 in 1000 patient exposures and less than 1 in 30,000 treatment sessions. In placebo controlled randomized trials of TMS for depression, TMS has been very well tolerated. In one clinical trial, the dropout rate for adverse events was 4.5% and adverse events were generally mild and limited to scalp discomfort (Breden Crouse, 2012). In another placebo controlled randomized trial, there were no statistically significant differences in adverse events between the active treatment arm and the sham arm for any parameter including headache, scalp discomfort, insomnia, worsening depression or anxiety, gastrointestinal symptoms, fatigue, muscle aches, vertigo, skin pain, facial twitching or other (Wajdik et al, 2010). In a third placebo controlled randomized trial of TMS in depression, the only adverse event found to differ between TMS and placebo was application site (scalp) pain (5% vs 0%; p=0.02) with no statistical difference seen in all other parameters including headache, muscle twitching, back pain, anxiety, and insomnia. This trial did report a single case of device-related serious adverse event (seizure). This patient had a two minutes generalized seizure during TMS, was managed with several hours observation in the Emergency Department, released without medical intervention, and had no reported sequelae as a result of the event. This event was reported to the FDA. EEG, ECG and EMG d. Discomfort from EEG: EEG is a widely utilized technique. There are no published or known risks associated with the procedure. The EEG instruments only record the electrical activity of the cortex from the scalp; these instruments cannot send out any electrical current. EEG has been used in thousands of studies and we are unaware of any injuries to participants. The procedure may entail slight discomfort associated with the removal of the gel after the experiment. e. Discomfort from EMG and ECG: EMG as well as ECG are widely used techniques. There are no known risks associated with the procedures. EMG and ECG instruments only record the electrical activity of muscles (EMG) and the heart (ECG); these instruments cannot send out any electrical current. EMG and ECG has been used in thousands of studies and we are unaware of any injuries to participants. The procedure may entail slight discomfort associated with the removal of the electrodes. As with a band-aid, the adhesive tugs on skin and hair when removed. Additionally, participants may experience slight skin irritation or dryness associated with the sandpaper and isopropyl alcohol pretreatment. If this occurs, the participant will be provided with an ointment to reduce skin irritation. Biomedical IRB Application Instructions Page 28 f. Risks associated with behavioral testing. There is a small risk of the loss of confidentiality. To address this risk the alphanumeric code designating the subject’s data will only be linked to their name via a master sheet, kept under lock and key, where the consent form will also be kept. The only place a name is recorded is on the master sheet, on the consent form, and on the payment slip to get reimbursement for participation. There is also a potential for boredom or fatigue. In order to minimize any discomfort, breaks are placed between blocks in the experiment. e. Risk of loss of confidentiality: There is a small risk that information about the identity of the subjects may be compromised, in this study. Subjects’ personal, identifiable information will be completely removed from any data associated with the individual. Questionnaire, behavioral and EEG data will be assigned a coded number. The identifiable information is only accessible by a restricted number of UCSD personnel at the PrTMS Research Center who has access to the key of the coding system. All identifiable information will be completely removed from data received by the PI and project associates at SCCN, UCSD. 15. RISK MANAGEMENT PROCEDURES AND ADEQUACY OF RESOURCES a. Discomfort of TMS TMS can cause brief activation of face or arm muscles. These twitches are not painful, but can feel odd or disquieting when first experienced. We will closely monitor subjects' comfort and will request them to tell us if the stimulation ever feels painful. If this occurs, we will immediately decrease TMS intensity to a level that is not painful. We will also offer participants acetaminophen (Tylenol) or aspirin before the rTMS session. ● In the case of neck stiffness or neck pain, participants will be offered acetaminophen (Tylenol) or aspirin which in most cases promptly resolves the discomfort. The experimental procedure will be immediately terminated whenever anyone reports experiencing discomfort. ● Related to the hearing issue, all volunteers will wear either earplugs or headphones (through which white noise will be played). The experimenters will also be provided with ear protection devices to minimize any discomfort they might experience from the clicks. ● Given that undergoing TMS can be a stressful experience for some subjects, especially in the first few minutes (while they get used to it), special care will be taken by the experimenter to check with the subject, at the outset (during the consenting process), that the subject is well rested and feeling healthy. b. Contraindications of TMS • Some subjects who are uncomfortable with being maintained in close quarters (i.e. within an experiment requiring them to keep their heads still) may experience the feeling to be trapped and could experience fainting/syncope during the TMS procedure. We will therefore exclude any subjects that have experienced claustrophobia previously or fainting due to the feeling of being trapped. If a participant reports feeling trapped anytime during the experiment we will immediately terminate the experiment. • TMS induces small electric currents, so metal anywhere in the head such as shrapnel, surgical clips or implanted devices such as cardiac pacemakers, aneurysm clips, cochlear implants, medical pumps, deep brain stimulators, or intracardiac lines are a contraindication. If a TMS coil is discharged close to the implanted wires connecting the electrodes to the implanted device, potentially significant voltages and currents could be induced, which could cause unintended neural stimulation and may present a safety risk. We will therefore exclude any subjects that have implanted devices or metal anywhere in the head. Standard tooth fillings are fine. • There is no known risk of TMS to pregnant women (either the mother or unborn foetus); however, the safety information of the of MagVita TMS Therapy system states that its safety and effectiveness has not been established in persons who are pregnant or nursing, therefore we will exclude this population. Biomedical IRB Application Instructions Page 29 Participants will be asked in the first screening inclusion/exclusion questionnaire whether they are pregnant. In addition all women in childbearing age will be offered a pregnancy test during the consent procedure right before the EEG/TMS session. She will be asked to take the test before starting the experiment. The experimenter will administer the test. If the test is positive or if she declines to take the test she will be excluded from the experiment ● There is a small risk of seizure induction by rTMS (see next point c. Risk of seizure induction or syncope). This will be more likely in people who have a neurological condition such as epilepsy, dementia, motor disorders, head injury or brain injury, therefore we will exclude this population. Participants are screened for these exclusion criteria during the recruitment process and again during the consent procedure right before the experiment. • People who are taking any psychiatric or neuroactive medications such as antipsychotics, mood stabilizers or anticonvulsants may have an increased risk for seizure induction. We will therefore exclude this population. Participants are screened for these exclusion criteria during the recruitment process and again during the consent procedure right before the experiment. • To avoid risk of thermal injury during rTMS we will cool down the coil before the experiment and in between experimental blocks. In case the coil heats up over 30 degree Celsius (the temperature of the coil can be monitored directly on the MagVenture TMS machine) we will immediately stop the stimulation. We will also exclude participants from the experiment that have a condition of Diabetes mellitus, a history of stroke or if participants currently use any sleep medication. These conditions may cause an impaired ability to sense heat/pain and may pose an increased risk of thermal injury during rTMS. c. Risk of seizure induction and syncope There is no need to take any special seizure precautions with TMS outside of staff training regarding the proper first responder actions for seizure. The following statement appears in the Clinical TMS Society consensus document (Perera et al, 2016): It is the consensus of the cTMSs that IV access, cardiac defibrillators, suction, and oxygen are NOT necessary for the safe administration of TMS in an outpatient TMS office. According to the consensus paper on the safety of TMS (Rossi et al., 2009) for research studies in normal subjects with conventional rTMS above 1Hz a non-medical setting is allowable (i.e. psychology lab, robotics lab, research institutions etc.) and the presence of a physician may not be required (see Table 12). Table 12: Uses of TMS and settings allowable (Rossi et al, 2009) This risk of seizure will be mitigated by: Biomedical IRB Application Instructions Page 30 ● ● ● ● ● Comprehensive safety screening of all participants, see attached list Having all personnel that will perform rTMS treatments (named as part of this proposal) trained in CPR and seizure management. We have also instituted the guidelines of a recent report (Rossi et al., 2009) to the effect that “Each TMS laboratory must institute an explicit plan for dealing with syncope and seizures, and every member of the TMS team must be familiar with it. There must be a place where the subject can lie down. All team members must be familiar with the means of summoning emergency medical help and when to call for it.”. We have such a place for subjects to lie down. We have posted in the TMS room clear instructions for how to call for medical help. All co-investigators are trained in CPR. Response to medical emergencies: The UCSD PrTMS Research Center located at 16918 Dove Canyon Rd, Suite 100, San Diego, CA 92127 will only perform rTMS experimental protocols at times when nursing or physician staff is on site to respond to the rare medical emergency. All staff managing and treating patients with TMS, technicians, nurses, and physicians, have specific training in the management of seizure and syncope emergencies including Basic Life Support (BLS). The community emergency medical system (EMS) will be activated for any seizure activity and EMS commonly responds to other medical emergencies in the same treatment building (e.g. chemotherapy infusion suite location on the same floor). Qualifications of TMS staff: The 2016 Clinical TMS Society Consensus Review and Treatment Recommendations for TMS (Perera et al., 2016) gives specific recommendations regarding the training of TMS staff. The Clinical TMS Society recommends that physicians and staff receive: a. Device specific industry-sponsored training AND one of the following: b. Additional training through a university affiliated or industry independent CME program OR c. Additional peer-to-peer direct supervision The 2017 Consensus Recommendations from the American Psychiatric Association, APA, (Perera et al, 2016) also give recommendations regarding training and credentialing the TMS team. The American Psychiatric Association recommends that rTMS prescribers be clinicians with prescriptive privileges who are knowledgeable about, trained, and credentialed in rTMS. They describe such training as including proficiency in all aspects of the rTMS procedure and recommend that each service develop its own policy regarding how many times a prescriber must obtain motor threshold or treat a patient before recredentialing of that prescriber (Perera et al., 2016). All staff managing participants with TMS will receive specific training on the MagVenture TMS Stimulation. Such training will be documented for each individual. Dr. David Hoopes, MD has received additional peer-to-peer direct supervision of TMS management with Dr. Kevin Murphy acting as the peer supervisor. Dr. Murphy has personally managed more than 1,000 TMS patients. A case-log of patients managed is maintained. Either Dr.Hoopes or Dr. Murphy will be on site (in the same building) at all times during experiments with TMS stimulation, to be able to respond to medical emergencies in the case of adverse events. The 2017 Consensus Recommendations from the APA (Perera et al., 2016) state that TMS operators should be trained in assessing motor threshold and administering TMS treatment. Examples of TMS operators in the APA recommendations include certified medical assistants, medical technicians with relevant experience, physician assistants, and nurses. In addition, all staff managing and treating patients with TMS, technicians, nurses, and physicians, will have specific training in the management of seizure and syncope emergencies including Basic Life Biomedical IRB Application Instructions Page 31 Support (BLS). documented. The TMS and BLS training of all staff, physicians and TMS operators, will be d. Discomfort of EEG and ECG Prior to the start of the experiment, subjects will be shown the EEG, EMG and ECG equipment. If any cognitive, emotional or physical symptoms appear to manifest or are mentioned by the subject, staff associates monitoring the situation will terminate the experiment immediately and reverse any potentially adverse effects if possible. e. Loss of confidentiality Participants will be recruited by CITI certified staff at the SCCN, UCSD. CITI certified UCSD personnel will deidentify EEG data, behavioral data and questionnaire data by assigning a coded number that is utilized for all EEG data collection and analysis purposes. The subject’s name, age, gender and the coded number will be kept in a password save folder at the PrTMS Research Center and only be linked to the EEG and behavioral data via a master sheet therein. No one except a restricted number of UCSD personnel at the PrTMS Research Center knows the subject’s name and/or his or her other identifiable information. For analysis the de-identified data will then be copied to the SCCN server that is password-protected at the Super Computer Center at UCSD. In the extremely unlikely chance that the password to the server is compromised, there is no risk of loss of confidentiality since recordings will not be linked with a subject’s name or any other identifying personal information. Screening will be performed via email by the investigator or CITI certified UCSD personnel at the SCCN and at the PrTMS Research Center listed on the research plan (and to be hired in the next month), but different than the person who administers the rTMS treatment. All participants will receive a code under which all data collected in the study (without any identifying information) is being stored. The code and the name of participants will be coded only in a master sheet that is stored in a separate place from the data in a password save folder on the server of the PrTMS Research Center. All consent forms, in the form of hard copy, will be stored in a locked cabinet inside the SCCN at UCSD that only the P.I. and staff associates have access to. EEG and clinical data on the SCCN server cannot be linked to consent forms since all subjects’ personal, identifiable information will be completely removed from any data associated with the individual. That identifiable information is only accessible by the UCSD personnel at the PrTMS Research Center. In the extremely unlikely chance that the password to the account at the PrTMS Research Center containing the Master Sheet that links personal information to the assigned numerical code mentioned above is compromised, we will immediately notify the individuals of the extent of their personal information that may have been released to unauthorized individuals. We will also attempt to rectify the situation by immediately recovering the information from public access and request that the recipient of said compromised information relinquish all copies to us. When the subject is asked to complete a behavioral questionnaire, the subject will also be informed that they have the right to stop the inventory at any time and may skip any questions that he/she is not comfortable answering. The results of the behavioral questionnaires will not be discussed with the subject. 16. PRIVACY AND CONFIDENTIALITY CONSIDERATIONS INCLUDING DATA ACCESS AND MANAGEMENT Biomedical IRB Application Instructions Page 32 All data recordings, will be labeled with the same de-identified coded number. After data is recorded on a local computer (e.g. data recorded on a hard drive of a dedicated recording computer at the PrTMS Research Center), it will be uploaded to the SCCN server at the Supercomputer Center at UCSD that can only be accessed by members of the SCCN lab. To be a member of SCCN, one must first obtain a username and password from our IT administrator. The IT administrator is only allowed to give these out once our P.I. has given permission. No one besides SCCN lab members will have access to de-identified data. Once again, subjects’ personal, identifiable information will be completely removed from any data associated with the individual before transfer to SCCN. That identifiable information is only accessible by the CITI certified UCSD personnel at the PrTMS Research Center. The study does not have foreseeable reason to report to any federal, state, or local authorities any of the data that is to be collected. 17. POTENTIAL BENEFITS There are no direct benefits to the subjects for participation in this. Subjects will be made aware that even though there is no direct benefit to them personally, their participation may bring insights into the brain mechanisms underlying rTMS brain stimulation, and may help to improve rTMS therapy in the future. 18. RISK/BENEFIT RATIO As outlined in section 9, paragraph ‘The safety of rTMS’ the range of stimulation parameters in the current protocol and inclusion criteria for participants all lie within the safety guidelines recommended by the FDA and and published literature. The trains used in this protocol last only 2-3 seconds and are therefore in a safe range (maximum safe train duration is >5 seconds for stimulation at 10 Hz with intensity at 110% of RMT). Participants will undergo a total of 5 minutes of rTMS in each experiment. Note that the intensity of the stimulation in our protocol is in the lower range - at 40-110 % of motor threshold. In addition, we will exclude participants if they have any personal or family history of epilepsy or neural disorders. We also exclude participants between 18-22 years old since in the MagVenture safety recommendation this group has been identified as a group with increased risk factor. In summary, in this protocol we adhere to all safety measures recommended for rTMS. The procedure therefore represents a safe procedure with minimal side effects. On the other hand a great deal of scientifically and clinically valuable information may be obtained from this study. The experiments proposed herein can provide new insights into rTMS induced changes on cortical oscillatory and excitability states and their impact on clinical improvements. Such results could have important implications for validating and improving rTMS treatments. Hence the possible benefits to this research exceed the risks. 19. EXPENSE TO PARTICIPANT No expense to the subjects, other than the time taken to complete the tests. 20. COMPENSATION FOR PARTICIPATION Subjects are reimbursed for their participation at the rate of $15.00 per hour, on a pro rata basis, including waiting time (e.g. when EEG is being setup). A typical experiment will last between 90 and 130 minutes all told depending on the study. 21. PRIVILEGES/CERTIFICATIONS/LICENSES AND RESEARCH TEAM RESPONSIBILITIES Biomedical IRB Application Instructions Page 33 The Principal Investigator of the study is Dr. Scott Makeig Ph.D. Director of the Swartz Center for Computational Neuroscience, UCSD. Dr Makeig is a Ph.D. cognitive neuroscientist with long expertise in electrophysiology and source analysis of EEG. Research will be conducted under his direction. UCSD faculty member David Hoopes, MD (Associate Professor, RMAS) will serve as one of the Co-PI on this protocol. He is physically located on site and will provide daily physician oversight of this protocol. Dr. Mateusz Gola is a clinical psychologist and project scientist at UCSD, and will also serve as a Co-PI on this protocol. He will provide oversight of this protocol and monitor possible assessments and exclusion of participants based on prior neurological or psychiatric disorders. Dr. Kevin Murphy, MD (is a physician at the Radiation Medicine and Applied Sciences Department at UCSD and has a private clinic where he treats patients with TMS in the same building as the PrTMS Research Center). Dr. Murphy will serve as a supervising physician in case of any adverse events during the TMS experiments. Either Dr.Hoopes or Dr. Murphy will be on site (in the same building) at all times during experiments with TMS stimulation, to be able to respond to medical emergencies in the case of adverse events. All staff managing rTMS stimulation protocols, technicians, nurses, and physicians, have specific training in the management of seizure and syncope emergencies including Basic Life Support (BLS). All named individuals below hold current CITI training certifications in Protection of Human Subjects (UCSD), and Research Aspects of HIPAA (UCSD). All individuals who undertake the TMS experimenter role are/will be trained in CPR and seizure management, and have certification. Certified staff to perform the rTMS stimulation and for recruiting participants will be hired in the next month by UCSD. Before any new personnel will have any contact with research subjects we will ask for an amendment to this IRB protocol to include these persons. 1. Dr. David Hoopes (MD) (Radiation Medicine and Applied Sciences, UCSD) is an associate professor and radiation oncologist at UCSD who will serve as the Co-PI in this study and oversee rTMS experiments under this protocol 2. Dr. Mateusz Gola (INC, UCSD) is a clinical psychologist and project scientist who will participate in the collection and analysis of data under this protocol 3. Dr. Johanna Wagner (INC, UCSD) is a post-doc who will participate in the collection and analysis of data under this protocol 4. Dr. Scott Makeig (INC, UCSD), the Director of the Swartz Center for Computational Neuroscience and PI in this project, will participate in the data analysis and interpretation. 5. Dr. Tzyy-Ping Jung (INC, UCSD) is a senior member of the lab running and analyzing EEG experiments. He will participate in the analysis of data collected under this protocol. 6. Luca Pion-Tonachini (INC, UCSD) is a graduate student who will participate in the collection and analysis of data under this protocol. 7. Sheng-Hsiou Hsu (INC, UCSD) is a graduate student who will participate in the collection and analysis of data under this protocol. 8. Clement Lee (INC, UCSD) is a lab technician who will participate in the collection and analysis of data collected 9. Dr. Yu-Te Wang (INC, UCSD) is a post-doc who will participate in the collection and analysis of data collected 10. Susana Brugues (INC, UCSD) is a research assistant who will participate in the collection and analysis of data collected. 11. Gail Garcellano (UCSD) is a clinical research nurse who will participate in the collection of data. 12. Dr. Kevin Murphy (MD) (Radiation Medicine and Applied Sciences, UCSD) is a radiation oncologist at UCSD who will oversee rTMS experiments under this protocol to provide medical assistance in the case of adverse events related to TMS. Biomedical IRB Application Instructions Page 34 22. BIBLIOGRAPHY (1) Bell, AJ, & Sejnowski, TJ. (1995). An information-maximization approach to blind separation and blind deconvolution. Neural Comput, 7:1129-59. (2) Breden Crouse, E. L. (2012). Transcranial magnetic stimulation for major depressive disorder: What a pharmacist should know. Mental Health Clinician, 2(6), 152-155. (3) Delorme, A., Mullen, T., Kothe, C., Acar, Z. A., Bigdely-Shamlo, N., Vankov, A., & Makeig, S. (2011). EEGLAB, SIFT, NFT, BCILAB, and ERICA: new tools for advanced EEG processing. Computational intelligence and neuroscience, 2011, 10. (4) Ding, L., Shou, G., Yuan, H., Urbano, D., & Cha, Y. H. (2014). Lasting modulation effects of rTMS on neural activity and connectivity as revealed by resting-state EEG. IEEE Transactions on Biomedical Engineering, 61(7), 2070-2080. (5) Fitzgerald, P. B., Maller, J. J., Hoy, K. E., Thomson, R., & Daskalakis, Z. J. (2009). Exploring the optimal site for the localization of dorsolateral prefrontal cortex in brain stimulation experiments. Brain stimulation, 2(4), 234-237. (6) Fröhlich, F. (2015). Experiments and models of cortical oscillations as a target for noninvasive brain stimulation. Progress in brain research, 222, 41-73. (7) Foa, E. B., Huppert, J. D., Leiberg, S., Langner, R., Kichic, R., Hajcak, G., & Salkovskis, P. M. (2002). The Obsessive-Compulsive Inventory: development and validation of a short version. Psychological assessment, 14(4), 485. (8) Fuggetta, G., Pavone, E. F., Fiaschi, A., & Manganotti, P. (2008). Acute modulation of cortical oscillatory activities during short trains of high-frequency repetitive transcranial magnetic stimulation of the human motor cortex: A combined EEG and TMS study. Human brain mapping, 29(1), 1-13. (9) Fuggetta, G., & Noh, N. A. (2013). A neurophysiological insight into the potential link between transcranial magnetic stimulation, thalamocortical dysrhythmia and neuropsychiatric disorders. Experimental neurology, 245, 87-95. (10) Hamidi, M., Slagter, H. A., Tononi, G., & Postle, B. R. (2009). Repetitive transcranial magnetic stimulation affects behavior by biasing endogenous cortical oscillations. Frontiers in integrative neuroscience, 3, 14. (11) Jin, Y., Potkin, S. G., Kemp, A. S., Huerta, S. T., Alva, G., Thai, T. M. & Bunney Jr, W. E. (2005). Therapeutic effects of individualized alpha frequency transcranial magnetic stimulation (αTMS) on the negative symptoms of schizophrenia. Schizophrenia bulletin, 32(3), 556-561. (12) Klimesch, W., Sauseng, P., & Gerloff, C. (2003). Enhancing cognitive performance with repetitive transcranial magnetic stimulation at human individual alpha frequency. European Journal of Neuroscience, 17(5), 1129-1133. (13) Makeig, S, Bell, AJ, Jung, TP, Sejnowski, TJ. (1996). Independent Component Analysis of electroencephalographic data. In: D. Touretzky, M. Mozer, and M Hasselmo (Eds), Advances in Neural Information Processing Systems 8, 145-151. (14) Makeig, S, Westerfield, M, Jung, TP, Enghoff, S, Townsend, J, Courchesne, E, Sejnowski, TJ. (2002). Dynamic brain sources of visual evoked responses. Science, 295: 690-693. (15) O’Reardon, J. P., Solvason, H. B., Janicak, P. G., Sampson, S., Isenberg, K. E., Nahas, Z.& Demitrack, M. A. (2007). Efficacy and safety of transcranial magnetic stimulation in the acute treatment of major depression: a multisite randomized controlled trial. Biological psychiatry, 62(11), 1208-1216. Biomedical IRB Application Instructions Page 35 (16) Perera, T., George, M. S., Grammer, G., Janicak, P. G., Pascual-Leone, A., & Wirecki, T. S. (2016). The clinical TMS society consensus review and treatment recommendations for TMS therapy for major depressive disorder. Brain stimulation, 9(3), 336-346. (17) Ridding, M. C., & Rothwell, J. C. (2007). Is there a future for therapeutic use of transcranial magnetic stimulation?. Nature Reviews Neuroscience, 8(7), 559-567. (18) Rossi, S., Hallett, M., Rossini, P. M., Pascual-Leone, A., & Safety of TMS Consensus Group. (2009). Safety, ethical considerations, and application guidelines for the use of transcranial magnetic stimulation in clinical practice and research. Clinical neurophysiology, 120(12), 2008-2039. (19) Thut, G., & Pascual-Leone, A. (2010). A review of combined TMS-EEG studies to characterize lasting effects of repetitive TMS and assess their usefulness in cognitive and clinical neuroscience. Brain topography, 22(4), 219. (20) Thut, G., & Miniussi, C. (2009). New insights into rhythmic brain activity from TMS–EEG studies. Trends in cognitive sciences, 13(4), 182-189. (21) Thut, G., Veniero, D., Romei, V., Miniussi, C., Schyns, P., & Gross, J. (2011). Rhythmic TMS causes local entrainment of natural oscillatory signatures. Current biology, 21(14), 1176-1185. (22) Veniero, D., Brignani, D., Thut, G., & Miniussi, C. (2011). Alpha-generation as basic response-signature to transcranial magnetic stimulation (TMS) targeting the human resting motor cortex: A TMS/EEG co-registration study. Psychophysiology, 48(10), 1381-1389. (23) Voigt, J., Carpenter, L., & Leuchter, A. (2017). Cost effectiveness analysis comparing repetitive transcranial magnetic stimulation to antidepressant medications after a first treatment failure for major depressive disorder in newly diagnosed patients–A lifetime analysis. PLoS one, 12(10), e0186950. (24) Wajdik, C., Roy-Byrne, P., Anderson, B., Nahas, Z., Bulow, P., Zarkowski, P., Holtzheimer, P.E., Schwartz, T., Lechner, T., Walch, T. and Deisenhammer, E.A., 2010. 1: Garcia-Toro M, Salva J, Daumal J, Andres J, Romera M, Lafau O, Echevarría M, Mestre M, Bosch C, Collado C, Ibarra O & Aguirre I. (2006) High (20-Hz) and low (1-Hz) frequency transcranial magnetic stimulation as adjuvant treatment in medication-resistant depression. Psychiatry Res.; 146 (1): 53-7. Psychiatry, 67(5), pp.507-16. (25) Walz, L. C., Nauta, M. H., & aan het Rot, M. (2014). Experience sampling and ecological momentary assessment for studying the daily lives of patients with anxiety disorders: A systematic review. Journal of anxiety disorders, 28(8), 925-937. (26) Wassermann, E. M. (1998). Risk and safety of repetitive transcranial magnetic stimulation: report and suggested guidelines from the International Workshop on the Safety of Repetitive Transcranial Magnetic Stimulation, June 5–7, 1996. Electroencephalography and Clinical Neurophysiology/Evoked Potentials Section, 108(1), 1-16. (27) Akalin Acar, Z., Acar, C., Makeig, S. (2016). “Simultaneous head tissue conductivity and independent EEG source location estimation.” NeuroImage, 124: 168-180. 23. FUNDING SUPPORT FOR THIS STUDY This is not an industry-sponsored study. This project is currently sponsored by a gift to the UC San Diego Foundation # 2467 by sponsor Kreutzkamp, and by a gift from the Swartz Foundation (Old Field, NY) 24. BIOLOGICAL MATERIALS TRANSFER AGREEMENT N/A Biomedical IRB Application Instructions Page 36 25. INVESTIGATIONAL DRUG FACT SHEET AND IND/IDE HOLDER N/A 26. IMPACT ON STAFF N/A 27. CONFLICT OF INTEREST N/A 28. SUPPLEMENTAL INSTRUCTIONS FOR CANCER-RELATED STUDIES N/A 29. OTHER APPROVALS/REGULATED MATERIALS N/A 30. PROCEDURES FOR SURROGATE CONSENT AND/OR DECISIONAL CAPACITY ASSESSMENT N/A Biomedical IRB Application Instructions Page 37